Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.)

Abstract

Induced mutagenesis finds a significant place in crop enhancement methodologies for bringing genetic variability in desired genetic backgrounds. Mutation breeding along with recombination breeding has developed >3300 mutant varieties in various crop species globally. In groundnut, radiation and chemical mutagenesis have been extensively employed for genetic improvement of vegetative, reproductive, agronomical, biochemical and physiological traits. Consequently, these mutant traits were instrumental in delivering 112 suitable productive cultivars in this allotetraploid leguminous crop. Induced mutants and their derived varieties acted not only as basic genetic pool for evolving desirable varieties but also for understanding various functions at biochemical and molecular levels. Numerous farmers, traders and exporters have benefitted by cultivating groundnut mutant varieties in many countries. Recent advances in genomics have facilitated to utilize molecular tools like gene editing, TILLING and mutagenomics for developing desired and improved traits in groundnut, addressing famers’ concerns, consumer preference and industrial needs.KeywordsInduced mutagenesisGamma raysEMSRecombination breedingMutant varietiesTILLINGMutagenomicsGene editing

Full text

SuprasannaPenna
S.MohanJainEditors
Mutation Breeding
forSustainable
Food Production
and Climate
Resilience

Editors
Suprasanna Penna
Nuclear Agriculture and Biotechnology
Division
Bhabha Atomic Research Centre
Mumbai, India
Amity Centre for Nuclear
Biotechnology & Amity Institute
of Biotechnology
Amity University
Mumbai, Maharashtra, India
S. Mohan Jain
Department Agricultural Sciences
University of Helsinki
Helsinki, Finland
ISBN 978-981-16-9719-7 ISBN 978-981-16-9720-3 (eBook)
https://doi.org/10.1007/978-981-16-9720-3
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487
Advances in Mutation Breeding
of Groundnut (Arachis hypogaea L.) 16
Anand M. Badigannavar and Suvendu Mondal
Abstract
Induced mutagenesis finds a significant place in crop enhancement
methodologies for bringing genetic variability in desired genetic backgrounds.
Mutation breeding along with recombination breeding has developed >3300
mutant varieties in various crop species globally. In groundnut, radiation and
chemical mutagenesis have been extensively employed for genetic improvement
of vegetative, reproductive, agronomical, biochemical and physiological traits.
Consequently, these mutant traits were instrumental in delivering 112 suitable
productive cultivars in this allotetraploid leguminous crop. Induced mutants and
their derived varieties acted not only as basic genetic pool for evolving desirable
varieties but also for understanding various functions at biochemical and molec-
ular levels. Numerous farmers, traders and exporters have benefitted by
cultivating groundnut mutant varieties in many countries. Recent advances in
genomics have facilitated to utilize molecular tools like gene editing, TILLING
and mutagenomics for developing desired and improved traits in groundnut,
addressing famers’concerns, consumer preference and industrial needs.
Keywords
Induced mutagenesis · Gamma rays · EMS · Recombination breeding · Mutant
varieties · TILLING · Mutagenomics · Gene editing
A. M. Badigannavar (✉) · S. Mondal
Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India
e-mail: anandmb@barc.gov.in
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2023
S. Penna, S. M. Jain (eds.), Mutation Breeding for Sustainable Food Production
and Climate Resilience,https://doi.org/10.1007/978-981-16-9720-3_16

488 A. M. Badigannavar and S. Mondal
16.1 Introduction
Groundnut (Arachis hypogaea L.) is an important edible oilseed, food and feed crop
cultivated globally on 27.52 million ha with a production of 45.52 million metric
tonnes (MMT), with a productivity of 1654 kg/ha during 2016–2020 period
(FAOSTAT 2020). Groundnut is the 2nd most important grain legume crop, 4th
most important oilseed crop and 13th most important food crop and is currently
cultivated in over 80 countries. Among the groundnut-growing countries, China has
the highest production (16.70 MMT) followed by India (7.50 MMT), Nigeria
(3.15 MMT) and the USA (2.68 MMT). It covers around 19.23% of the cultivated
area with 27.33% contribution to the total oilseed production in India (ICAR-IASRI
2019). It is distinct from the rest of the legume species by having above-ground
flower and below-ground fruit. Fruit development involves aerial elongation, below-
ground pod expansion, seed filling and finally seed drying.
Groundnut is mainly consumed for cooking oil, protein, minerals and vitamins in
many nations and contributes considerably to food security in turn alleviating
poverty. Around 50% of the total global groundnut produce is utilized for oil
purpose; 35% is for food or confectionary purpose, and the remaining 15% is for
seed and animal feed (Birthal et al. 2010). Groundnut oil is primarily used for
cooking and manufacture of soap and margarine. Seeds are consumed as raw or
roasted nuts, in making confectioneries or butter. Tender pods are also eaten as
vegetable. Protein-rich de-fatted cake is a nutritious livestock feed. Microbe-
processed groundnut shells and cakes are used to manufacture commercially essen-
tial enzymes.
Groundnuts have recently been considered as a functional food. Several studies
revealed that its consumption has distinct advantages on human health. Diets
enriched with groundnut and its butter reduced the risk of heart disease, cancer,
total cholesterol, bad cholesterol and triglycerides without affecting beneficial cho-
lesterol due to the presence of monounsaturated fatty acid, resveratrol, beta-
sitosterol, vitamin E, folic acid and fibre (Francisco and Resurreccion 2008). It
contains nearly half of the minerals and vitamins necessary for normal body growth
and maintenance. Additionally, groundnut skins or its extracts are identified as an
antioxidant, antimicrobial agent, functional food and animal feed component
(Toomer 2020). Due to its satiety value, it prolongs hunger and is helpful in dieting
for body weight maintenance. Children gained body weight and height with ground-
nut protein fortifications.
Genetic variability for a wide array of characters is of utmost necessity for plant
breeding success. In nature, mutations, though their frequency is very low, are the
main source of variability. Breeder with the help of ionizing radiations and some
mutagenic chemicals enhances the mutation frequency. Mutation breeding involves
induction of genetic variability and employing induced variability either directly or
in cross-breeding. With the specific objectives and targeting, improvement of one or
two traits in a well-adapted variety is fundamental to success in mutation breeding at
many institutes including ours. Sustained efforts in mutation breeding for many

decades resulted in commercial release of >3300 crop varieties in different species
worldwide (IAEA 2021).
16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 489
Cultivated groundnut (A. hypogaea L.) exhibits narrow genetic base, regardless
of having considerable agronomical, morphological and physiological variability
because of its restricted gene flow due to ploidy barrier, self-pollination and mono-
phyletic origin (Holbrook and Stalker 2003). Due to paucity of genetic variability,
groundnut is vulnerable to many biotic and abiotic stresses. Breeding for better pod
yield continues to be the primary objective in any of groundnut improvement
activities. Induced mutagenesis is one of the convenient and desirable approach
for broadening genetic variability to overcome the limitations associated with a
narrow genetic basis and could bring specific improvement without significantly
affecting other traits in groundnut (Patil and Mouli 1979). Extent of induced
variability is dependent on mutagen type, studied character and genetic background
(Murty et al. 2004; Nadaf et al. 2009; Badigannavar et al. 2020). Induced mutants
not only serve as parental material in crop breeding but are also genetic resources for
functional genomics. In groundnut, effective application of induced mutagenesis
along with cross-breeding has led to the generation of a wide spectrum of mutants for
plant type, early maturity, pod type and size, seed size and composition, testa colour
and tolerance to both biotic and abiotic stresses in groundnut (Patil and Mouli 1979;
Mouli et al. 1989a; Kale et al. 2000a; Mondal et al. 2007; Gowda et al. 2010; Kavera
et al. 2013; Yu et al. 2019; Badigannavar et al. 2020; Brown et al. 2021). This review
envisages experiences and recent developments in mutation breeding in groundnut.
16.2 Mutagens
In groundnut mutation breeding experiments, the objectives are to induce mutants
with better pod yield, ideal plant type, desirable pod traits, high shelling percentage,
large seed, moderate seed dormancy, early maturity, high oil content, improved seed
nutritional traits and tolerance to diseases, salinity, moisture stress, heat, etc. Many
of the physical and chemical mutagens were effectively employed to induce desir-
able mutants, in turn leading to mutant varieties (Table 16.1). In early years, mainly
X-rays and later gamma rays were used for induced mutagenesis. Additionally, fast
neutrons, electron beam and heavy-ion beam have been used for mutation induction
in groundnut. Electron beam irradiation was standardized using linear accelerator
(10 MeV) for low-dose (50–1000 Gy) application for groundnut in India (Mondal
et al. 2017). Based on the electron beam treatment of seeds of five groundnut
genotypes, GR
50
(50% growth reduction) values ranged from 108 Gy in TG
51 (most radio-sensitive) to 270 Gy in TAG 24 (most radio-tolerant). For gamma
rays, corresponding values were 295 Gy and 385 Gy, respectively. Another ionizing
radiation with a high linear energy transfer (LET), fast-neutron irradiation, can bring
secondary ionization as well as gene mutations in plant cell, resulting in stable
mutant traits (Wang et al. 2015). Heavy-ion beams, due to their greater LET and
relative biological effect (RBE) as compared to gamma rays or X-rays, induce
greater mutation rate, in turn generating a wide range of mutation types. X-rays

yield low-LET radiation (0.2 and 5 keV/μm) resulting in few ionizations. Hence,
most probably, low-LET radiation will mainly cause easily repairable single-strand
DNA breaks. On the contrary, heavy ions and fast neutrons having higher LET
induce clusters of double-strand breaks in the DNA, which are difficult to repair
(Shikazono et al. 2005; Cabanos et al. 2012). The effective dose for gamma rays
ranges from 200 to 350 Gy, which is close to GR
50
depending on the genotype and
radio-sensitivity factors (Badigannavar and Murty 2007; Nadaf et al. 2009).
Decreased germination and increased seedling mortality, pollen sterility, and mor-
phological and cytological anomalies were reported with the increasing doses of
490 A. M. Badigannavar and S. Mondal
Table 16.1 Different mutagens used for induced mutagenesis in groundnut
Mutagen Dose/concentration References
Physical mutagens
X-rays 10–35 kR Gregory (1955), Bora et al. (1961), Patil and
Bora (1961), Patil (1968), Menon et al.
(1970), Patil and Mouli (1977)
Gamma rays 100–600 Gy Mouli and Patil (1976), Reddy et al. (1977),
Mouli and Kale (1982b), Pathirana et al.
(1998), Branch (2002), Liu et al. (2004),
Badigannavar and Murty (2007), Mondal
et al. (2007), Nadaf et al. (2009), Hassan and
Anes (2015), Badigannavar et al. (2020), Fu
et al. (2021)
Electron beam 150–250 Gy Mondal et al. (2017)
Fast neutron 10–18 Gy Wang et al. (2015)
Neutron beam 2.5 ×10
12
–5×10
13
n/
cm
2
s
Shivraj et al. (1962), Shivraj and Ramana
Rao (1963)
Heavy-ion beam
(C, N)
100 Gy Cabanos et al. (2012)
Laser Liu et al. (2004), Lin et al. (2005)
Chemical mutagens
Ethyl
methanesulphonate
(EMS)
0.01–0.5% Ashri and Levy (1976), Prasad et al. (1984),
Sivaram et al. (1989), Gowda et al. (1996),
Mathur et al. (2000), Nadaf et al. (2009),
Wang et al. (2011b), Chen et al. (2020)
Diethyl sulphate
(DES)
0.01–0.2% Ashri and Goldin (1965), Ashri and Levy
(1976), Mathur et al. (2000)
EMS + DES 0.01–0.1% + 0.01–0.1% Mathur et al. (2000)
Ethidium bromide 500 ppm Levy and Ashri (1978)
Acriflavine 1.15 mM Ashri et al. (1977)
Sodium azide 3 mM; 0.41% for 2 h Prasad et al. (1985), Mathur et al. (1998),
Mondal et al. (2007), Wang et al. (2002,
2011a), Nkuna et al. (2021)
Nitroso methyl urea 0.02% Prasad et al. (1984)
Pingyangmycin 4 mg/L Sui et al. (2015)

gamma rays or concentrations of ethyl methanesulphonate (EMS) (Gowda et al.
1996; Badigannavar and Murty 2007; Mondal et al. 2007). Mutagenic effectiveness
was higher in gamma rays than the EMS, while with an increased rate of dose/
concentration, mutagenic efficiency was decreased for lethality and pollen sterility
and increased for injury (Manjunath et al. 2020). With an increase in doses of fast
neutrons, there was decrease in the frequency of somatic embryo formation and
plantlet regeneration (Wang et al. 2015).Mathur et al. (1998) standardized the LD
50
dose of 0.41% for a 2-h treatment for sodium azide in groundnut. Subsequently,
Mondal et al. (2007) found that the combination of sodium azide and gamma rays
was ideal for developing groundnut mutants with greater genetic variability.
16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 491
Seeds (M
0
) of groundnut cultivars, mutants, selections, advanced lines or hybrids
are usually treated with various mutagens (Murty et al. 2004). Subsequently, treated
seeds (M
1
) are sown in the field. Usually, desirable mutants are selected from M
2
generation onwards, and their breeding behaviour is confirmed in the next
generations. Since then, usual breeding methods like pedigree method, bulk method
or single-seed descent for agronomic evaluation have been followed. The induced
mutants are exploited in recombination breeding by crossing mutant with mutant,
mutant derivative, breeding line or cultivar and mutant derivative with cultivar
(Fig. 16.1).
16.3 Cytogenetic Aberrations
Groundnut is an allotetraploid (2n=4x=40, AABB) legume crop belonging to the
Fabaceae family. Radiations are known to induce chromosomal aberrations in the
M
1
generation. The meiotic aberrations due to X-ray irradiation of groundnut
cv. Spanish Improved were irregular development of spindles and their activity,
abnormal association of chromosomes and their separation at anaphase, occurrence
of bridges and fragments, failure of cytokinesis, formation of polyads and anomalies
in the development of pollen grains (Bora et al. 1961). Further, many reciprocal
translocations leading to the formation of chain and ring multivalents, inversions
resulting in fragments and bridges at anaphase I and II and persistent anaphase I
bridges at telophase II were reported (Patil and Bora 1961). In M
4
progenies, Patil
(1968) observed trisomics, tetrasomics, cytomixis, long chromosomes with dis-
turbed coiling mechanism and 15–18 chromosomes in pollen mother cells. Ashri
et al. (1977) induced three trisomic mutations by treating developing embryos with
acriflavine. X-ray irradiation of inter-specific(A. hypogaea ×A. monticola) hybrid
showed occurrence of monosomics (Menon et al. 1970). Asynaptic chromosomes
with very low frequency of bivalents at diakinesis were observed in the X-ray-
induced dwarf mutant (Patil and Mouli 1977). An altered chromosomal organization
in meiotic prophase and early metaphase stages, a spherical dot instead of normal
round centromere in meiotic metaphase and the displaced spindle fibres in most
stages of meiosis were observed (Hassan and Anes 2015). Many chromosomal
aberration types like earlier separated mitosis metaphase, polypolarity division,

492 A. M. Badigannavar and S. Mondal
Fig. 16.1 Evolution of groundnut mutant and mutant-derived varieties developed at Bhabha
Atomic Research Centre, Mumbai, India (red square), and their usage as parents for mutant variety
development by state agricultural universities (blue square), which are released and notified across
the country

sticky, non-equal division and nucleus protrusion into cytoplasm were also found
due to laser irradiation (Lin et al. 2005).
16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 493
Recently, sequential genomic in situ hybridization (GISH) and fluorescence in
situ hybridization (FISH) were employed to study chromosomal variations in Chi-
nese variety, Silihong, and in 70 gamma ray-induced M
1
plants with two multiplex
probe cocktails, total genomic DNAs of A. ipaensis and A. duranensis, and 45S and
5S rDNAs as the probes (Fu et al. 2021). In 14 M
1
plants, 8 monosomic
chromosomes, 1 deletion and 17 translocations were identified. In one of the M
1
plants, one reciprocal translocation between chromosomes 1A and 3B was also
observed. In these chromosomal changes, nine translocations were observed
between non-homologous chromosomes and eight translocations between
homoeologous chromosomes. Higher number of translocations were seen in
chromosomes 1, 3 and 5. Such chromosomal variations in groundnut could be
employed for gene mapping, radiation hybrid mapping and translocation or deletion
mapping.
16.4 Mutations for Seed Size
In groundnut, large seeds attract greater consumer and market preference for confec-
tionery and value addition purpose, which may get better price in domestic and
international markets. Groundnut growers often face problems with the large-seed
varieties like late maturity, low shelling outturn, less proportion of sound mature
kernels, longer seed dormancy and lower yields. Induced mutagenesis was success-
fully employed for the development of desirable high-yielding large-seed varieties
with earliness and optimum seed dormancy, making them fit for diverse cropping
patterns. In late 1950s, X-ray treatment of cv. Spanish Improved induced a large-
seed mutant, which was later released as TG 1 based on its superior yield perfor-
mance (Patil 1975) (Fig. 16.1). Inter-mutant cross between TG 1 and dark-leaf
mutant resulted in extreme fastigiata, high-yielding variety, TG 17 (Patil 1977).
Subsequently, by including TG 1 and TG 17 in crosses, TG 22 with medium-large
seeds and TKG 19A and Somnath with large-seed varieties were developed and
released (Mouli et al. 1989b,1990). Further, a large-seed sister line of TKG 19A, TG
19, was recombined with TAG-24 to evolve three high-yielding large-seed varieties,
TG 39, TLG 45 and RARST-1 (TG 47) with 115–120 days to maturity, 72–75%
shelling outturn, 70–80 g HKW (hundred kernel weight) and higher proportion of
large seeds (Kale et al. 2008,2010;Badigannavar et al. 2012). Towards diversifica-
tion of large-seed genetic base, gamma ray mutagenesis of TAG 24 (Badigannavar
and Murty 2007), TG 38 (Badigannavar et al. 2020) and TG 66 (Mondal et al. 2007)
and electron beam mutagenesis of TG 26 (Mondal et al. 2017) were carried out
periodically and several high-yielding large-seed (HKW: 70–80 g) mutants (TG 77,
TG 78, TG 79, TG 89, TG 90) with 110–115 days to maturity were isolated, which
are performing better in national and state evaluation trials. Similarly in Sri Lanka,
Pathirana et al. (1998)isolated three high-yielding gamma ray-induced mutants with
large seeds. Branch (2002) selected four mutants having larger pod and seed and

greater percentage of extra-large kernels than parent through gamma ray mutagene-
sis. Field performance of one mutant showed 23% higher pod yield than parent.
Further, sodium azide treatment of cv. L7-1 induced mutants with a greater change in
pod and seed size and weight (Wang et al. 2002). Joshua and Bhatia (1983)
attributed increased seed size in groundnut to the increased cotyledonary cell volume
by retaining similar cell number within the unit area.
494 A. M. Badigannavar and S. Mondal
16.5 Mutations for Early Maturity
Early-maturing groundnut genotypes facilitate in escaping end-season drought and
end-season rains and fit into different cropping systems like paddy fallows, residual
moisture situation and post-potato crop. Early maturity also makes farmers to harvest
early and bring the produce early to the market, which in turn fetch better price.
Breeding for earliness in groundnut is complicated due to its indeterminate nature
and subterranean cumulative process of pod maturation. By exercising selection
pressure for better pod yield, higher shelling outturn and greater HKW from early
harvest, early-maturing (95 days) mutant derivatives TGE-1 and TGE-2 were
evolved by recombining mutants (Patil et al. 1982; Mouli and Kale 1982a)
(Fig. 16.1). The maturity was further brought down to 90 days with desirable traits
using induced mutagenesis (Mouli and Kale 1989). Early maturity and high yielding
ability were brought together in TAG-24 (Patil et al. 1995). Further, earliness from
Chico was combined with better pod yield of TG 26, leading to the development of a
new variety, TG 51, having 90 days to maturity, greater pod yield and better shelling
(Kale et al. 2009). Gamma ray mutagenesis of TG 51 along with polyethylene glycol
(PEG)-based screening for moisture-stress tolerance has identified drought-tolerant,
early-maturing mutant, TG 84, which has shown better performance for seed yield in
national evaluation trials.
16.6 Mutations for Subspecific Traits
In Arachis genera, 81 species including the cultivated groundnut, A. hypogaea, have
been reported (Valls et al. 2013). A. hypogaea is categorized into subspecies,
fastigiata and hypogaea, which are further classified based on morphological
features into botanical varieties. Accordingly, ssp. fastigiata is classified into var.
aequatoriana, var. fastigiata (Valencia), var. peruviana and var. vulgaris (Spanish),
while ssp. hypogaea into var. hirsute (Peruvian runner) and var. hypogaea (Virginia
runner). Consistent generation of mutations and recombination in groundnut resulted
in interchanges for intra-specific and inter-specific characters among genotypes
shown in Tables 16.2 and 16.3.

–
16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 495
Table 16.2 Studies on mutations between groundnut botanical varieties
From To
Variety Spanish
bunch (var.
vulgaris)
Valencia
(var.
fastigiata)
Virginia bunch
(var.
hypogaea)
Virginia runner
(var.
hypogaea)
Spanish bunch
(var. vulgaris)
–7 1, 7, 8 2, 3, 6, 7
Valencia (var.
fastigiata)
7–2, 7 7
Virginia bunch
(var.
hypogaea)
4, 5, 7 –7
Virginia runner
(var.
hypogaea)
3, 7 7 7 –
References: 1. Patil (1966); 2. Reddy et al. (1977); 3. Levy and Ashri (1978); 4. Mouli and Kale
(1982a); 5. Mouli et al. (1986); 6. Hussein et al. (1991); 7. Gowda et al. (1996); 8. Mondal et al.
(2007)
Table 16.3 Mutant or mutant derivatives showing mutations at subspecies levels in groundnut
Traits Mutant or mutant derivatives References
fastigiata genotypes with
hypogaea traits
BARCG 1, BARCG 2, TG
7, TG 8, TG 9, TG 10, TG
12, TG 13, TG 13A, TG 16, TG
17, TG 18A, TKG 19A, TG
22, TG 23, TAG 24, TG 25, TG
26, TG 27, TG 28, TG 28A, TG
40, TPG 41, TG 42, TG 44, TG
45, TG 46, TG 47, TG 48, TG
49, TG 77, TG 78, TG 79, TG
89, TG 90
Mouli and Kale (1982b), Mouli
and Kale (1989), Mouli et al.
(1989a,1990), Kale et al.
(2000b), Badigannavar and
Murty (2007), Mondal et al.
(2007,2017), Badigannavar
et al. (2020)
hypogaea genotypes with
fastigiata traits
TG 1A, TG 18, TG 39, TG 43 Kale et al. (2000b)
Genotypes having
hypogaea spreading habit
and sequential flowering
TGS-1 (Somnath), TGS-2 and
TGS-3
Mouli et al. (1989b)
Genotypes with one or
more modified characters
absent in fastigiata and
hypogaea
TGE-1, sl,sl-imp mutants, TG
18A-84, TG 21
Mouli and Kale (1982a), Patil
and Mouli (1984)
16.7 Mutations for Trait Association
Many of the groundnut traits are associated and inherited together in succeeding
generations. Such associations are disintegrated due to induced mutagenesis
resulting in favourable recombinants. The usual negative correlation between seed
size and oil content or shelling outturn or maturity was disassociated through

induced mutants or inter-mutant crosses, leading to genotypes with large seeds
having high oil content, early maturity and increased shelling outturn (Patil 1973;
Mondal et al. 2017). High water-use efficiency and high harvest index, otherwise
negatively correlated, were recorded in inter-mutant derivatives, TAG-24 and
Somnath, in multi-location trials (Murty et al. 2004).
496 A. M. Badigannavar and S. Mondal
16.8 Mutations for Biotic and Abiotic Stress Tolerance
In groundnut, biotic and abiotic stresses severely affect the seed productivity and
quality. The induced mutants and their derivatives tolerant to diseases, pests,
drought, acid soil, heat and cold have been reported in groundnut (Table 16.4).
Taxonomically important groundnut genotype, Dharwad Early Runner (DER) upon
successive EMS treatments, resulted in induced mutants (mutants 1-45, 1-110, 28-2)
Table 16.4 Mutant or mutant derivatives of groundnut showing tolerance to biotic and abiotic
stresses
Trait Tolerant mutant or mutant derivatives References
Late leaf Dh 232, GPBD 5, 1-45, 1-110, Mutant Mathur et al. (2000), Motagi et al.
spot 28-2, PBS 30107, PBS 30108 (1996,2022)
Early leaf
spot
ICGV 76, GG 13 Basu (2002)
Rust Dh 232, GG 13, GPBD 5, PBS 30108,
PBS 30138
Mathur et al. (2000), Basu (2002),
Motagi et al. (2022)
Collar rot TG 37A NRCG (2000)
Peanut bud
necrosis
disease
TAG 24, TG 37A, R 9251 Patil et al. (1995), Giriraj and Itnal
(1999), NRCG (2000)
Peanut stem
necrosis
disease
TKG 19A NRCG (2000)
Spodoptera
litura
Mutant 28-1, 28-2, 45, 110-1 Rajendra Prasad et al. (1998)
Thrips Mutant 28-1, 28-2, 45, 110, 172 Rajendra Prasad et al. (1998)
Bruchid
beetle
TSP 60, PBS 30001 Mathur et al. (2000)
Phosphorus
efficiency
PBS 30016, PBS 30026 Mathur et al. (2000)
Acid soils ICGV 76, TKG 19A Annual report (2001)
Salinity Binachinabadam-5, Binachinabadam-6 Azad et al. (2014)
Drought Dh 256, GG 35, PBS 30008, PBS
30022, PBS 30023, PBS 30109, Tafra-
1, TG 37A, TG 39, TPT 25
Mathur et al. (2000), Kale et al.
(2004a,2010), Abdalla et al. (2018),
Motagi et al. (2022)
Heat PBS 30109, PBS 30138, TG 18 Mathur et al. (2000), Anonymous
(2002)
Cold TAG 24 Dave and Mitra (2000)

with late leaf spot (LLS) resistance (Motagi et al. 1996). Further, some of these
mutants showed resistance to Spodoptera litura (mutants 28-1, 28-2, 45, 110-1) and
to thrips (mutants 28-1, 28-2, 45, 110, 172) (Rajendra Prasad et al. 1998). EMS
mutagenesis of mutant VL1 having rust resistance and LLS susceptibility produced
morphologically similar mutants with LLS resistance and rust susceptibility (Gowda
et al. 2010). Origin of resistant mutant was later related to the insertion of
A. hypogaea miniature inverted-repeat transposable element (AhMITE1) at FST1-
linked site. Subsequent spontaneous mutation in these mutants produced variants
which were LLS and rust susceptible. Origin of such variants was linked to the
excision of AhMITE1 from FST1-linked site, demonstrating its main role in high-
frequency origin of LLS-resistant mutants in groundnut. Towards developing
aflatoxigenic resistant fungal mutants in groundnut, Azzam et al. (2007) isolated
mutants with significant decrease in occurrence of Aspergillus flavus and
A. parasiticus and aflatoxin in comparison to parent, Giza 5. These mutants com-
pared to Giza 5 had the lowest content of aflatoxin B1 and/or B2 under soil
infestation with aflatoxin-producing fungi and were found free from aflatoxin
contamination under field conditions.
16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 497
Salinity, being an important abiotic stress, impacts various growth stages of
groundnut including final yield. It is ideal to develop salinity-tolerant groundnut
genotypes to minimize yield losses due to salinity problem. So far in groundnut,
tangible results have not been achieved due to lack of simple and suitable screening
method for salinity tolerance in large populations. Towards this aspect, a simple
screening protocol for larger segregating or mutant populations for radicle growth
was developed (Badigannavar et al. 2007). Considerable genetic variability for
germination under 400 mM NaCl stress was noted in established gamma ray mutants
of TAG 24. In six of the TAG 24 mutants, greater radicle growth was noted by
screening at 125 and 150 mM NaCl. Further, three mutants at 125 mM and one
mutant at 150 mM NaCl had better tolerance due to longer radicle length and lesser
radicle reduction. Further, TPG 41, a large-seed variety, was irradiated with gamma
rays to induce genetic variability for radicle growth under salinity (Badigannavar
et al. 2007). By screening large number of seeds at 100 mM NaCl in the M
3
followed
by screening plant-wise in the M
4
and M
5
, 91 true breeding mutants with salinity
tolerance for radicle growth were obtained. Ahmed and Mohamed (2009) identified
three salinity-tolerant mutants obtained through gamma ray and sodium azide
treatment when they were evaluated with soil and irrigation water salinity (ECe)
having 13.15 dS/m and 6.5 dS/m, respectively, in M
5
and M
6
generations. Mutants
produced higher seed and pod yield per plant and more number of pods and seeds per
plant than their parents. Azad et al. (2014)studied groundnut mutants in the
Bangladesh coastal belt under saline field conditions. It was found that the high-
yielding mutants under rainfed condition mostly took a lesser period to close the
stomata in spite of their higher stomatal and lower cuticular transpiration rates. Of
these, two mutants having comparatively better yield than ‘Dacca-1’were
commercialized as salt-tolerant varieties by names ‘Binachinabadam-5’and
‘Binachinabadam-6’.

498 A. M. Badigannavar and S. Mondal
In vitro mutagenesis of Huayu 20 seeds with pingyangmycin and screening on a
medium having hydroxyproline was carried out to obtain drought-tolerant mutants
(Sui et al. 2015). The seedlings of M
3
individuals were screened for drought stress.
The activities of superoxide dismutase and peroxidase were substantively raised in
eight progenies of hydroxyproline-tolerant, regenerated plants than parent. Addi-
tionally, under drought stress, few M
3
progenies gave more pods compared to
parent. Abdalla et al. (2018) evaluated groundnut mutants for end-season drought
tolerance in Sudan. By using the rainout shelter, 25 days’terminal drought was
imposed after 60 days from planting. These terminal drought-survived mutants were
further evaluated for yield performance under rainfed situation. The mutant
Barberton-B-30-3 gave a mean pod yield of 1024 kg/ha as compared to 926 kg in
check variety over 12 seasons. Further, the stability and GGE biplot analysis also
showed that Barberton-B-30-3 was more stable by producing better yield in both low
and high rainfall seasons and was officially released as ‘Tafra-1’in 2018.
16.9 Mutations for Physiological Traits
Though the physiological traits are genetically quantitative in nature, few studies
have shown mutations for such traits in groundnut. Chlorophyll synthesis was
studied in wild-type and virescent mutant leaves by continuous illumination of
dark-grown seedlings (Benedict and Ketring 1972). Chlorophyll synthesis in the
virescent leaves showed a 72-h lag period compared to the wild-type leaves before
the onset of rapid chlorophyll accumulation. The development of chloroplast grana,
protein synthesis and activity of many enzymes of the malate dehydrogenase,
phosphoenolpyruvate carboxylase and reductive pentose phosphate cycle decreased
in the virescent leaves during the lag phase of chlorophyll accumulation. Later,
Alberte et al. (1976) compared the photosynthetic activity and chloroplast lamellar
system of wild-type and virescent leaves. The leaves in mutant have shown 42%
reduction in chlorophyll, a reduction in the number of photosystem I reaction centre,
a higher chlorophyll a/b ratio and a change in proportions of the two chlorophyll-
protein complexes of the chloroplast. In addition, the mutant had 1.5 times larger
photosynthetic unit size than the wild type. Groundnut mutants, TG 1 and TG
16, showed higher photosynthetic rate than their parent Spanish Improved. Among
the two, TG 1 was found more efficient. The translocation of photosynthates to
nodules was also greater in TG 1. Comparatively higher nitrogen fixation and dark
CO
2
fixation capacity in nodules were reported in the same mutants (Lodha et al.
1983,1985).
16.10 Mutations for Seed Biochemical Traits
Induced mutagenesis has played a vital role in altering seed nutritional traits like oil
content, fatty acid, protein, minerals and vitamins. Seed oil content was increased by
2% from 49.5% in the mutants of groundnut cv. JL-24 (Mouli et al. 1987). Inter-

mutant crosses resulted in high oil (55%) genotypes with a 5% increase (Patil 1973).
On the contrary, a mutation also brought down oil content from 46% to 38% (Mouli
and Kale 1991). Doo et al. (2008) observed 43.2–53.5% oil among gamma ray
mutants compared to 47.8% in parent Shinnamkwang. Similarly, oil content was
enhanced by 4.3–6.1% in seven gamma ray mutants of TAG 24 along with better
seed and oil yield (Badigannavar and Mondal 2009). With an in vitro mutagenesis of
Huayu 20 seeds with pingyangmycin, mutant seeds had >57% oil as compared to
53.7% in parent (Sui et al. 2015). Subsequently, three mutants with oil contents
57.7%, 61.1% and 59.3% were released as new groundnut varieties, Yuhua 4, Yuhua
9 and Yuhua 14, respectively (Wang et al. 2020).
16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 499
Groundnut seeds with higher oleic acid have shown greater shelf life, and their
consumption has several advantages to human health (Kris-Etherton et al. 2001;
Moreira et al. 2014). Mutations affecting modified oleic and linoleic acid (O/L)
ratios were obtained in induced TG mutant and mutant derivatives (3.16–3.32) as
compared to parent, Spanish Improved (1.02) (Sharma et al. 1981,1985). Mondal
and Badigannavar (2010) isolated gamma ray mutant TGM 71 from large-seed
variety, TPG 41, with higher oleic acid (68.7%) and lower linoleic acid (13.6%),
in turn enhancing O/L ratio to 5.0 as compared to its parent (61.9%, 19.3%, 3.2,
respectively). Another mutant, TGM 192M obtained from gamma rays and sodium
azide treatment, showed better oleic acid (70.7%) and lesser linoleic acid (12.8%)
than its parent, TFDRG 5 (55.3%; 26.0%), which in turn increased the O/L ratio to
5.5 from 2.0 (Mondal et al. 2011). Further, this high oleate mutant was improved for
pod yield by hybridizing it with the varieties, TPG 41 and TG 51, and directed
selection resulted in eight high-yielding advanced selections with 70–75% oleic acid
(Mondal et al. 2018; Badigannavar et al. 2020). In addition to gamma rays, EMS
mutagenesis also generated a narrow-leaf high-oleate mutant (68.6%) from TMV
2 (38.5%) (Prasad et al. 1984; Mondal and Badigannavar 2013). An inter-specific
disease-resistant variety, GPBD-4, with both EMS and gamma ray mutagenesis
resulted in several mutants with >70% oleic acid as compared to 50.7% in parent
(Kavera et al. 2013). Recently, a normal oleic cv., Fuhua 12, was successively
treated with EMS in two rounds, and mutants having 76.9–83.9% oleic acid were
isolated in contrast to 35.9% in their parent (Yu et al. 2019).
Induced mutations have contributed to protein enhancement in groundnut.
Induced mutants, TG 1 (27.68%) and TG 16 (31.64%), were reported to have higher
protein compared to parent, Spanish Improved (13.59%) (Sharma et al. 1981,1985).
These mutants also had higher contents of histidine, lysine, phenylalanine, proline
and tryptophan and lower contents of methionine, serine and threonine. The limiting
amino acids were tryptophan in Spanish Improved; threonine in TG 8, TG 9 and TG
17; and valine in TG 18. Further, the essential amino acid content per seed excepting
methionine, valine and threonine was higher in all the mutants. Doo et al. (2008)
found 23.3–31.7% protein in gamma ray mutants compared to 26.8% in parent
Shinnamkwang. An EMS-derived mutant having 28.6% protein was isolated as
compared to 17.7% in its parent (Wang et al. 2013). A high-seed-protein mutant
TGM 206 (31%) with selective increase in conarachin fraction was identified from
gamma ray mutagenesis of TG 66 (26%) (Mondal and Badigannavar 2016). In an

in vitro mutagenesis of Huayu 20 with pingyangmycin followed by screening on a
medium with hydroxyproline, mutant seeds contained higher protein (>30%) than
parent (26.6%) (Sui et al. 2015). Some of the TG varieties showed enhanced sucrose
content. Large-seed mutant derivative like TKG-19A contained higher sucrose,
lower raffinose and stachyose and desirable nutritional traits for ‘table purpose’
(Gadgil and Mitra 1982,1983). Sucrose content among gamma ray mutants ranged
from 2.6% to 6.2% compared to 4.5% in parent Shinnamkwang (Doo et al. 2008).
500 A. M. Badigannavar and S. Mondal
Heavy-ion (C or N) beam irradiation was employed to develop 17 knockout
hypoallergenic groundnut mutants from the Japanese Nakateyutaka variety
(Cabanos et al. 2012). Of these, eight mutants lacked either one of the two isoforms
of Ara h 2 or other nine mutants lacked one of the isoforms of Ara h 3. Wan et al.
(2016) noted lower level of anthocyanin, lignin and proanthocyanidin and higher
level of melanin in mutants having seed coat crack as compared to wild type. In
white-seed-coat mutant, lower anthocyanins and higher isoflavones and flavones
were recorded as compared to its parent (Wan et al. 2020).
16.11 Mutation Breeding for Climate Resilience in Groundnut
Climate change due to addition of greenhouse gases to the atmosphere brings more
frequent high/low temperatures, droughts, floods, cyclones, hailstorms and persis-
tent sea-level rise, which are expected to pose a severe threat to agriculture, biodi-
versity and human society (Karavolias et al. 2021). Climate change along with
abiotic stresses would also increase disease and pest incidence by exposing crops
to enhanced biotic pressure. To cope with these disasters and to tackle various biotic
and abiotic stresses, it is crucial to breed crop varieties with climate resilience.
Mutation breeding has the potential to evolve climate-resilient genotypes in various
crops including groundnut, in turn moderating the effects of climate change on
agriculture.
Groundnut crop encounters frequent early, mid-season and terminal drought
stress especially in rainfed-based cultivation, which is the major area in African
and Asian countries. Groundnut mutant derivatives like TAG 24, TG 37A and TG
51 with semi-dwarf type, early vigour, better harvest index and assimilate
partitioning were reported to have enhanced moisture stress tolerance, and their
earliness assists them to escape end-season droughts (Murty et al. 2004;
Badigannavar et al. 2020). Recently, Abdalla et al. (2018) developed gamma
ray-induced end-of-season drought-tolerant groundnut mutants in Sudan. Another
mutant-derived variety, Dh 256, having mid-season drought tolerance has recorded
better pod yield and higher relative water content compared to check, G 2-52, under
water-stress condition (Motagi et al. 2022).
On the other hand, groundnut crop has been experiencing excessive rains at the
pod-filling/maturity stage in recent times due to shift in rainfall pattern. Such
excessive humid conditions are highly congenial for groundnut to get greater
prevalence of rust and leaf spot diseases affecting seed yield and quality

considerably. Towards this aspect, several late leaf spot-resistant groundnut mutants
were isolated through EMS mutagenesis (Motagi et al. 1996; Gowda et al. 2010).
16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 501
In recent times, larger groundnut area in India suffered from in situ seed germi-
nation due to end-season rains spoiling almost ready-to-harvest produce. Varieties
with optimum period of fresh seed dormancy would rescue the rain-induced seed
germination and spoilage. Hussein et al. (1991) induced gamma ray mutants from
non-dormant parent, Early Bunch, with 6–7 weeks of seed dormancy. Trombay
groundnut mutants or their derivatives like TG 1, TKG 19A, TG 22, TG 26 and TPG
41 with their 3–5 weeks’fresh seed dormancy are ideal to curtail the yield losses due
to in situ seed germination (Murty et al. 2004; Badigannavar et al. 2020).
16.12 Mutant Varieties
In groundnut, mutation breeding has played a significant role in enhancing produc-
tivity, which has been the main objective in majority of the country’s breeding
programmes. Selection was practised consistently for yield components like pod
number, seed size, shell thickness and branch number in different mutant
generations. Generally, most productive and widely adapted popular cultivar was
subjected for induced mutagenesis to derive mutant varieties. Usually, mutants have
been detected in the M
2
on a single-plant basis (Donini et al. 1984). In many
breeding experiments including at Trombay, mutants selected for academic interest
sometimes even with inferior agronomic traits had also served as good parents for
evolving agronomically superior breeding lines (Patil and Mouli 1979). Based on
available literature, around 112 groundnut varieties involving 46 direct mutants and
66 mutant derivatives have been released globally, and Bangladesh, China, India and
the USA are leading countries (Table 16.5).
Radiation experiment in groundnut was initiated way back in 1949 in the USA
(Gregory 1955). A high-yielding, disease-resistant mutant was developed by
irradiating cv. Virginia Bunch with X-rays and subsequent selection. Comparable
with the best varieties grown, the mutant was released as NC 4x in 1959, the first
groundnut mutant variety in the USA (Gregory 1960). GA-T2636M is another
induced mutant with a high O/L ratio derived from gamma irradiation of ‘Georgia
Runner’(Branch 2000). By involving GA-T2636M in crosses, two varieties,
Georgia Hi-O/L and Georgia-02C, and a breeding line, GA 942004, were developed.
Further, through GA 942004 in back crosses with Georgia Green, Georgia-09B was
developed. Subsequently, by incorporating high oleate trait from Georgia-02C,
Georgia-09B and Georgia Hi-O/L, another seven high-yielding varieties (Georgia-
08V, Georgia-10T, Georgia-12Y, Georgia-13M, Georgia-14N, Georgia-17SP,
Georgia-18RU) were developed in the USA (Table 16.5) (Brown et al. 2021).
Besides, high O/L cultivar C458 (Flavor Runner 458) was selected from
EMS-treated Florunner. In Argentina, an X-ray-induced mutant, Colorado Irradiado,
having high yield and oil content became a successful cultivar in the late 1970s
(Livore et al. 2018).

(continued)
502 A. M. Badigannavar and S. Mondal
Table 16.5 Groundnut mutant or mutant-derived varieties developed in different countries
Country/trait Direct mutant varieties Mutant-derived varieties
Argentina
(2)
a
High yield Colorado Irradiado
Large seed Virginia No. 3
Bangladesh
(9)
High yield Binachinabadam-1,
Binachinabadam-2,
Binachinabadam-4, BINA
Chinabadam-10
Large seed Binachinabadam-3
Salinity
tolerance
Binachinabadam-5,
Binachinabadam-6,
Binachinabadam-7,
Binachinabadam-9
China (40)
High yield Fu 21, Huayu 16, Lainong
10, Xianghuasheng 4, Yuhua
5, Yuhua 7
8130, 78961, Huayu 22, Huayu 32, Lu
8130, Luhua 13, P 12, Yangxuan
1, Yangxuan 58, Yueyou 22, Yueyou
33, Yueyou 551, Yueyou 551-6,
Yueyou 551-38, Yueyou 551-116,
Zhonghua 16
Large seed Huayu 40 Huayu 9610, Yueyou 169, Yueyou
187, Yueyou 187-93
Early
maturity
Ganhua 1, Luhua 6 Luhua 15, Xianghua 1
Low
temperature
resistance
Changhua 4
Large seed Luhua 7
Resistance to
Aspergillus
flavus
Fu 22
Rust
resistance
Shanyou 27, Yueyou 223
Drought
tolerance
Luhua 11
High oil Yuhua 4, Yuhua 9, Yuhua 14
India (41)
High yield CO 2, G 2-52, TG 3, TG 38, TAG 73 BSG 0912, Co (Gn)-5, Dh 40, Dh
86, Dharani, JCG 88, GG 21, GG
34, GG 41, PDKVG 335, RG 559-3,
TG 51, TCGS 894
Large seed BG 1, BG 2, TG 1 RARS-T-1 (TG 47), Somnath, TLG
45, VRI 2
Drought
tolerance
Dh 256, GG 35, TG 37A, TPT 25

16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 503
Table 16.5 (continued)
Country/trait Direct mutant varieties Mutant-derived varieties
Seed
dormancy
R 9251, TKG 19A, TG 22, TG 26
High harvest
index
JL 501, TG 17, TAG 24
Late leaf spot
and rust
resistance
GPBD 5, Dh 232
High oleic
acid
Dh 245 TG 39, TPG 41
Malaysia (2)
Early leaf
spot
resistance
KARISMA Serene
High vitamin
A and invert
sugar
KARISMA Sweet
Myanmar (1)
Early
maturity
Sin Padetha 1
Pakistan (1)
High yield,
red testa
Golden
Sri Lanka (1)
High yield,
early maturity
ANK-G1 (Tissa)
Sudan (1)
Drought
tolerance
Tafra-1
USA (12)
High yield NC 4x
High oleic
acid
C 458 Georgia Hi-O/L, Georgia-02C,
Georgia-08V, Georgia-09B, Georgia-
10T, Georgia-12Y, Georgia-13M,
Georgia-14N, Georgia-17SP, Georgia-
18RU
Viet Nam (2)
High yield B 5000, DT 332
Total (112) 46 66
a
Total number of mutant or mutant-derived varieties of the country
In India, groundnut improvement through radiation-based mutation breeding was
initiated during 1957–1958 at the Bhabha Atomic Research Centre, Trombay,
Mumbai, which evolved a robust mutant gene pool having distinct mutants and
mutant derivatives for developing varieties in subsequent years (Patil and Mouli
1979). In total, 41 varieties were developed by mutation breeding and have been

released for farmer cultivation in India since 1973 (Table 16.5). Of these, nine are
direct induced mutants and rest are all mutant derivatives. TG 1 (Trombay groundnut
1) was the first induced large-seed mutant variety developed in 1973 by X-ray
irradiation of Spanish Improved with alterations from var. vulgaris to var. hypogaea
(Patil 1966) (Fig. 16.1). TG 3, being another direct mutant variety, also showed
superior yield performance with greater number of tertiary branches (Patil 1966).
Interaction of two mutant genomes resulted in increase in oil content in TG 9 (Patil
1973), reduced number of branches (extreme fastigiata type) and increased pod yield
in TG 17 (Patil 1977). TG 1 along with TG 17 resulted in large-seed variety, TKG
19A. Two early-maturing selections, TGE 1 and TGE 2, were evolved as a result of
genomic blend of three mutants and mutant genome in the background of Gujarat
dwarf mutant, respectively. Under the different genomic backgrounds, suitable
mutant derivatives, Somnath (TGS 1) and TG 22, were developed to become
subsequently promising cultivars. Genomic blend of four mutants of Spanish
Improved under the M 13 background developed TAG 24 (Patil et al. 1995), TG
39 (Kale et al. 2010), TLG 45 (Kale et al. 2008) and RARST-1 (TG 47)
(Badigannavar et al. 2012) (Fig. 16.2). Similarly, one mutant of JL 24 and four
mutants of Spanish Improved along with M 13 resulted in yet another successful
cv. TG 26 (Kale et al. 1997). The genomic constitution of TG 26 was expanded with
natural mutant Gujarat dwarf, Girnar 1, and early genetic stock Chico, respectively,
to evolve three varieties TG 37A, TG 38 and TG 51 (Kale et al. 2004a,2007,2009)
(Fig. 16.2). Likewise, TPG 41 was developed from one mutant of JL 24 and four
mutants of Spanish Improved under Robut 33-1 and M 13 backgrounds (Kale et al.
2004b). Recently, a gamma ray mutant of TG 38, TAG 73, has been released. The
entire genetic base for TG germplasm was constituted by seven parents, viz., Chico,
Girnar, Gujarat dwarf, JL 24, M 13, Robut 33-1 and Spanish Improved (Fig. 16.1),
with induced mutants of Spanish Improved being the main contributors to TG
evolution (Badigannavar et al. 2002). TG varieties were also one or the other parent
in the development and release of another 14 varieties in various states (Fig. 16.1)
(Badigannavar et al. 2020). EMS mutagenesis of GPBD 4 also contributed to the
development of a promising variety G 2-52 (Nadaf et al. 2009) and Dh 245 (Nadaf
et al. 2017) and its derivatives, GPBD 5 and Dh 232 (Motagi et al. 2022).
504 A. M. Badigannavar and S. Mondal
Fig. 16.2 Some groundnut mutant or mutant-derived varieties in India: TAG 24, TG 37A, TG
39, TLG 45 and TG 51 (left to right)

16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 505
In China, mutation breeding was initiated in the early 1960s and applied success-
fully to develop improved groundnut varieties. As a result of intensive mutation
research in many institutes, around 40 mutant or mutant-derived varieties have been
released, which include 16 direct mutant varieties (Table 16.5). In the 1960s,
groundnut mutant Fushi was developed by irradiating cv. Shitouqi with beta rays
from
32
P. By using Fushi in cross-breeding, a first variety Yueyou 22 was developed
in 1968 followed by Yueyou 33 in 1971. Later, Yueyou 22 contributed as parent in
developing another six varieties (Fu 21, Yuexuan 58, Yueyou 551, Yueyou 551-6,
Yueyou 551-38, Yueyou 551-116) between 1972 and 1986. Most of the groundnut
varieties planted in Guangdong and South China provinces have a pedigree of
mutant Fushi. The popular, widely grown cv. Huayu 22 in northern China was
developed by gamma rays and hybridization (Wu et al. 2006). Mutation breeding has
also been successfully adopted for groundnut improvement in Uganda (Busolo-
Bulafu 1991) and in Egypt (Ahmed and Mohamed 2009).
16.13 Significance and Coverage of Groundnut Mutant
and Mutant-Derived Varieties
In China, numerous groundnut mutant varieties have been cultivated on larger area
bringing remarkable economic, social and ecological benefit and sustaining crop
production of the country (Liu et al. 2004). The cumulative area of Chinese mutant
cultivars covers about 20% of the total area under groundnut (Qiu et al. 1997). Series
of ‘Yueyou’varieties have reached the accumulated growing areas of more than
4 million ha. Cultivation of Yueyou 22 reached an area of 130,000 ha. Fu 21, a
gamma ray mutant of Yueyou 22, and Yueyou 33, a selection from Yueyou
22, covered an area of 11,000 ha and 20,000 ha, respectively. Yueyou 551 evolved
by crossing Yueyou 22 with Yueyou 431 was grown extensively on 222,000 ha in
Guangdong province. By involving Yueyou 551 in cross-breeding, five more
varieties, Yangxuan 1, Yuexuan 58, Yueyou 551-6, Yueyou 551-38 and Yueyou
551-116, were developed, and of these, Yueyou 551-116 became the most promi-
nent variety with 130,000 ha area coverage. Fushi-derived varieties had been grown
on 3 million ha in South China, and groundnut production was enhanced to the tune
of 500,000 tonnes with 500 million Yuan to the farmers (Jiang and Zhou 1987,
1988). In Argentina, an X-ray-induced mutant variety, Colorado Irradiado, occupied
around 80% of the groundnut area (262,000 ha) in the 1970s (Livore et al. 2018).
In India, mutation breeding has contributed notably for the development of large-
seed varieties with early maturity (TG 39, TPG 41, TLG 45, TG 47), which are
suitable for table purpose and export under bold types. Seeds of the varieties like TG
37A, TG 38 and TG 51 are preferred for the export under Java type. Semi-dwarf
habit, high harvest index and better partitioning in TAG 24, TG 26 and TG 47 permit
pegs to enter the soil early to have better and uniformly developed pods. The
compact plants of TAG 24, TG 26, TG 38 and TG 47 facilitate farmers for high-
density planting for attaining greater productivity. Fresh seed dormancy in TKG
19A, TG 22, TG 26 and TPG 41 prevents in situ seed germination of matured crop

due to end-season rains. Hence, this character is very beneficial under current
changing climatic conditions, wherein prolonged end-season rains are often experi-
enced. Early maturity in TAG 24, TG 26 and TG 51 is helpful to escape end-season
drought and to have groundnut crop in paddy fallows, residual moisture situation and
post-potato crop. Hence, farmers are able to earn greater income due to better price in
the market. Drought tolerance in TAG 24, TG 37A and TG 39 makes them suitable
for cultivation in water-limited areas. High oleic acid (60%) in TG 39 and TPG
41 imparts better oil shelf life and health benefits. TAG 24 is used as the national
check variety and TG 37A and TG 51 as zonal check varieties in national evaluation
trials.
506 A. M. Badigannavar and S. Mondal
Fig. 16.3 Field view of groundnut variety, TG 39 (left) along with farmer (right)
Widespread coverage of these TG varieties in major groundnut-growing Indian
states has benefitted thousands of farmers, traders and exporters. Farmers have been
experiencing the better yielding ability of TG varieties by harvesting the record
yields (>7000 kg/ha) apart from superior yields (3000–4000 kg/ha) obtained in
many parts of the country. Two progressive farmers from Maharashtra state
harvested record yields of 9280 kg/ha dry pods and 10,175 kg by cultivating TAG
24 and 9487 and 10,542 kg by growing TG 26 under suitable agro-ecology such as
balanced nutrition, uninterrupted but controlled irrigation in summer environment
and disease-free conditions (Kale et al. 2002). Another progressive farmer from
Andhra Pradesh obtained a record yield of 7800 kg pods/ha by cultivating TG
51 under irrigated conditions (DGR 2010). Of late, large-scale field demonstrations
of TG 37A in Maharashtra during 2019 and 2020 showed superiority of 37% in pod
yields and 71% in net returns over local variety, SB XI, to the farmers. Similarly, TG
37A also recorded 28% and 24% greater pod yield and net returns, respectively, in
Tamil Nadu. Recently, TG 39 has earned price advantage to the Gujarat farmers by
securing the highest market price (Fig. 16.3). Annual breeder seed demand for
mutant and mutant-derived varieties from the various states in India ranged from
10% to 30% of the total demand for all the varieties put together with an average of
18% (>145 tonnes) in the last two decades.

16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 507
16.14 TILLING in Groundnut
Targeting Induced Local Lesions in Genomes (TILLING) is a potent reverse genetic
tool to detect mutants in high-throughput and gene sequence-assisted manner. The
technique is equally applicable for all traits if the gene sequence is known. The
universality of TILLING made it popular in many crop species for detecting mutants
for agronomic, biochemical and stress-related traits. It involves PCR-based amplifi-
cation of the target genes, followed by heteroduplex formations and cleavage by
CEL1 nuclease or other endonucleases to identify single-nucleotide or small inser-
tion/deletion mutations. The technique has been applied in maize (Till et al. 2004),
rice (Till et al. 2007), soybean (Cooper et al. 2008) as well as groundnut (Knoll et al.
2011; Guo et al. 2015). It was a challenge to develop allergen-free groundnut for
human consumption. Knoll et al. (2011) addressed this problem by mutation induc-
tion and screening the mutant population through TILLING approach. Before the
genome sequence availability, Ara h2.01 and Ara h2.02 were cloned from cDNA
library by employing nested PCR. LICOR-gel-based screening of 3420 M
2
plants
from EMS-mutagenized population of Tifrunner and nine SNPs in Ara h 2.01,five in
Ara h 2.02 and four in Ara h 1.01 were identified. In Ara h 1.01,a‘C-to-T transition’
at 593 nucleotide position was silent, but the other three SNPs were predicted to
induce amino acid changes: R333W, P405L and E437K. The non-synonymous
mutation R333W at position 333 situated within epitope 12 of Ara h 1.01 (Shin
et al. 1998). Only one mutation (a premature stop codon) was confirmed in Ara
h 1.02 that resulted in a truncated protein (Knoll et al. 2011). TILLING was also used
to identify a frameshift mutation in AhFAD2B (Knoll et al. 2011). The overall
mutation frequency for EMS was found to be 1 SNP/967 kb in the above six
genes in groundnut.
Subsequently, the researchers investigated mutations for stress-related genes by
using a subset of 768 M
2
plants from the above mutant population of Tifrunner
through TILLING by sequencing (Guo et al. 2015). Phospholipase D (AhPLD) and
lipoxygenase 7 (AhLOX 7) genes were upregulated in pod in response to drought and
infection to Aspergillus spp., respectively. Using the above mutant population, four
missense mutations in AhLOX7 and three missense mutations in AhPLD were
detected. Further, one missense and two silent mutations for Ara h 1.01, three silent
and five missense mutations in Ara h 1.02, one silent mutation in Ara h 2.02 and one
missense mutation in AhFAD2B were also identified by following TILLING by
sequencing in this subset mutant population (Guo et al. 2015). It was revealed that
the SNP detection frequency for single-copy genes was 1 SNP/344 kb and
1 SNP/3028 kb for multiple-copy genes in groundnut. Recently, Karaman et al.
(2021)analysed EMS-induced mutant population by TILLING. The mutation in
ahFAD2B resulted in change from serine to threonine and from glycine to aspartic
acid in ahFAD2A. The estimated overall mutation rate was 1 mutation in every
2139 kb. The mutation frequencies were also 1/317 kb for ahFAD2A in 0.4% EMS
and 1/466 kb for ahFAD2B in 1.2% EMS treatments. Taking its genome size of
2800 Mbp (2800 ×1000 kbp), at least 2800 plants must be screened to get a mutation
in any gene through EMS mutagenesis.

508 A. M. Badigannavar and S. Mondal
16.15 Molecular Characterizations of Mutants Through Target
Gene-Based Approach
In groundnut, genes for oil accumulation in seed were studied by examining
transcriptome of developing seeds of a normal oil cultivar, Huayu 22 (49.5%), and
its high-oil-EMS mutant, O1 (60.9%), at 40 days and 47 days after flowering (DAF)
using GeneFishing technology (Tang et al. 2013). After cloning and sequencing of
distinguishable differentially expressed bands, 27 unigenes from 40 DAF and
13 from 47 DAF were identified. Homology search revealed that 20 unigenes
(17 from 40 DAF, 3 from 47 DAF) were highly homologous to known gene
sequences for oil biosynthesis, energy metabolism, signal transduction and stress
response. Of them, three differentially expressed genes encoding thioredoxin,
oleosin and transaldolase were further confirmed by real-time quantitative PCR.
By using induced mutagenesis with gamma rays and sodium azide in groundnut,
a high-oleic-acid (70%) mutant was isolated and later characterized through target
gene sequencing approach (Mondal et al. 2011). Sequencing of mutated ahFAD2A
gene detected two non-synonymous mutations in the coding region. Multiple
sequence alignments of the AhFAD2B gene from Huayu 22 (wild type) and high-
oleate mutant revealed a C281T transition in the coding region causing an I94T
(isoleucine to threonine at 94th amino acid position) substitution in the oleoyl-PC
desaturase (Wang et al. 2011a). A Virginia-type high-oleate (>60%) mutant (E2-4-
83-12) was isolated from an EMS treatment of LF2 (44.2% oleic acid). Cloning and
sequencing of AhFAD2B from LF2 and E2-4-83-12 identified a novel mutation,
C313T, in the coding region causing an H105Y non-synonymous substitution in the
first histidine box of the FAD2B protein (Fang et al. 2012). Two stable high-oleic-
acid (>70%) mutant lines, viz. GM6-1 and GM4-3, isolated from GPBD 4 were
utilized for characterization of AhFAD2B gene (Nadaf et al. 2017). Cloning and
sequencing of FAD2B gene from GPBD 4, GM6-1 and GM4-3 revealed two novel
mutations (A1085G and G1111A) in GM 6-1 and single transition (G1111A) in GM
4-3. A CAPS (bF19/R1, MobII enzyme) marker and two SNP markers (bF19/GM6-
1-GM4-3 and bF19/GM6-1) that could differentiate the two mutants were also
developed (Nadaf et al. 2017). Sequence comparison of the high-oleate EMS mutant
and parent Fuhua 12 revealed a G448A substitution in AhFAD2A and an A insertion
(441_442insA) in AhFAD2B genes, which together contributed to high oleate in
mutant (Yu et al. 2019). Zhuang et al. (2019) developed mutants Min6-A from EMS
treatment of Minhua 6 and Min8-A from gamma ray irradiation of Minhua 8 with
~80% oleic acid. Sequencing identified mutations in AhFAD2A (dysfunction muta-
tion on AH09G33970 at 114,779,221 bp of Chr09) and AhFAD2B (frameshift on
AH19G43590 at 154464257 bp of Chr19), which conferred high oleate trait.
Recently, Nkuna et al. (2021) identified a high-oleic Virginia-type groundnut mutant
from sodium azide mutagenesis. Sequencing of the mutated and wild-type FAD2A/
FAD2B genes detected two mutations, viz. the G448A mutation in FAD2A resulting
in an amino acid change of D150N and G558A in FAD2B, causing a stop codon and
premature termination of protein synthesis.

16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 509
16.16 Mutagenomics for Characterization of Mutants
Mutagenomics research in groundnut was initiated by Yu et al. (2015) through the
usage of GeneFishing technology. Using this technique, 40 differential transcripts
were identified between high-protein mutant and its parent. Of them, three unique
genes with nutrient reservoir activity, protein disulphide oxidoreductase activity and
ATPase/transporter activity, respectively, were related to high-protein phenotype in
the mutant.
Next-generation sequencing is a well-established, versatile genomics platform
with many applications in plant biology research. This technique along with mutants
and mutant-derived population is now being used in many mapping techniques like
MutMap (Abe et al. 2012), MutMap-Gap (Takagi et al. 2013a) and QTL-seq (Takagi
et al. 2013b) to know the nature of mutations and to identify mutant gene. Testa
colour in groundnut is an important quality attribute that determines consumer
preference. Using RNA sequencing approach, Wan et al. (2016) studied the differ-
ential transcript behaviour of a brown testa mutant with crack seed coat (pscb) and
wild-type seed coat harvested at 20, 40 and 60 DAF. By analysing gene expression
patterns and sequences of 62 differentially expressed genes (DEGs) between mutant
and wild type, three candidate genes, namely, c36498_g1 (CCoAOMT1; Caffeoyl-
CoA O-methyltransferase 1), c40902_g2 (kinesin) and c33560_g1 (MYB3), were
suggested to be responsible for seed coat cracking and brown colour phenotype.
Later, the same group of scientists worked towards identification of DEGs on a small
pod width mutant (pw) compared to its parent by using RNA-sequencing of devel-
oping pod samples at 20, 40 and 60 DAF. A genome-wide comparative analysis of
expression profiles revealed 260 DEGs across all three stages, and two candidate
genes, c26901_g1 (CAD; cinnamyl alcohol dehydrogenase) and c37339_g1 (ACS;
1-aminocyclopropane-1-carboxylate synthase), responsible for pod width were
identified (Wan et al. 2017). Recently, an integrative analysis of transcriptomes,
metabolomes and histocytology of white-seed-coat (wsc) mutant revealed that the
mutant gene influenced the flavonoid biosynthesis in testa as well as suberin
formation, glycolysis, TCA cycle and amino acid metabolism. Further, common
DEGs’analysis in the above three pod development stages detected three testa-
specific expressed candidate genes Araip.M7RY3,Aradu.R8PMF and Araip.
MHR6K that were likely responsible for the white testa in the mutant (Wan et al.
2020).
The same transcriptomic approach was used to characterize a susceptible late leaf
spot EMS mutant M14 of a resistant cultivar Yuanza 9102 (Han et al. 2017).
RNA-seq analysis in the leaf tissues of M14 and Yuanza 9102 under pathogen
challenge showed 2219 DEGs including 1317 up-regulated genes and 902 down-
regulated genes. DEG analysis revealed up-regulation of inducible pathogenesis-
related genes and down-regulation of genes related to photosynthesis in susceptible
M14 mutant. The study suggested that the up-regulation of WRKY transcription
factor along with depression of chloroplast genes and plant hormone-related genes
for plant growth happened in response to fungal infection in susceptible mutant that

resulted in reduction of photosynthesis and phytohormones and led to symptom
formation.
510 A. M. Badigannavar and S. Mondal
In another high-throughput transcriptome study on gamma ray-induced semi-
dwarf mutant 2 (sdm2), DEGs were found to be involved in cell wall synthesis and
metabolic pathways and hormone biosynthesis and signalling pathways (Guo et al.
2020). The expression of several genes in BR biosynthesis and signalling was found
to be considerably down-regulated in leaf and stem of sdm2 as compared to parent.
Further genes in cell wall synthesis and metabolic pathways, which are related to cell
elongation, were downregulated in the stem of sdm2 mutant. In the study of muta-
epigenomics, Bhat et al. (2020) identified differentially methylated sites in the
genome of an EMS mutant TMV2-NLM and its parent, TMV 2. This narrow leaf
mutant differed in 240 methylation sites in A genome and 401 methylation sites in B
genome. Of the 641 differentially methylated sites, only 45 were found in 37 genes
among which 8 had differential expression between mutant and parent. This study
first demonstrated the role of EMS mutagenesis in DNA methylation in groundnut.
16.17 Gene Editing for Site-Directed Mutagenesis
Recently, gene editing studies based on zinc finger nucleases (ZFN), single-stranded
oligonucleotides, transcription activator-like effector nucleases (TALENs) and clus-
tered regularly interspaced short palindromic repeats (CRISPR/Cas9) have been
reported in several polyploid crops (Kaur et al. 2018; Arndell et al. 2019). The
site-directed approach also allows selection of desirable plants by delivery of
biodegradable Cas9/sgRNA ribonucleoprotein complexes (no foreign DNA) into
plant cells, where they are acted transiently but allow for efficient gene editing (Woo
et al. 2015). Such a system has been demonstrated in Camelina sativa, an emerging
oil seed plant, wherein oleic acid was increased from 16% to >50% with a concomi-
tant decrease in linoleic acid and linolenic acid (Jiang et al. 2017). These innovative
systems creating precise mutations will be useful in enhancing oleate content even in
groundnut.
Much before the availability of complete genome sequence of tetraploid ground-
nut, scientists have explored the possibility to further improve the oil quality by
changing the oleic/linoleic (O/L) ratio in seed oil. Huang et al. (2008) generated
RNAi lines against FAD2 gene in groundnut and isolated transgenic plants with
higher O/L ratio than the wild type. After the detection of sequence variation in
natural and induced high-oleic mutants, researchers started to manipulate these
quality characters through site-directed mutagenesis approach. Wen et al. (2018)
attempted TALEN-mediated gene editing in both FAD2A and FAD2B genes of
cv. YueYou no. 7. Oleic acid level in transgenic seeds was increased up to
90.45% with the concomitant reduction of linoleic acid to 7.42%. This is the first
successful report on the use of TALEN in targeted mutagenesis of groundnut.
However, TALEN technique has its inherent constraints of target-specific TALE
protein construction in vector and low mutation frequency of T1 plants. Most of the
site-directed approach depends on transformation efficiency, which is still low in

groundnut compared to other crops. However, the genome editing efficiency in
groundnut can be enhanced by other potential sequence-specific nucleases, including
ZFNs, CRISPR/Cas9 and CRISPR/Cpf1 systems (Lowder et al. 2016). CRISPR/
Cas9-mediated site-directed gene editing was used to mutate FAD2 gene by both
protoplast and hairy root transformation methods in groundnut (Yuan et al. 2019).
They had identified the hotspot site (called as sgRNA6 site) within FAD2 that
showed higher frequency of ‘G448A’and ‘G451T’transition in AhFAD2A and
AhFAD2B, respectively. In addition, ‘441_442 insertion A’in FAD2B similar to
natural mutation in F435 was also identified in this study. The mutation ‘G451T’in
AhFAD2B was novel and generated a premature stop codon. Such site-specific
mutagenesis using CRISPR-Cas9 is equally important to know the function of
uncharacterized gene in groundnut. On this direction, Shu et al. (2020) used this
technology in groundnut and found that AhNFR5 gene played a major role in nodule
formation. Application of site-directed mutagenesis technique in studying the role of
genes or creating valuable traits in groundnut has enough scope in post-genomic era
due to the availability of information on gene families, copy number and sequence
divergence within homologues. Such information will help to design more specific
sgRNA construct, which will selectively mutate the homologue in polyploid crop
like groundnut.
16 Advances in Mutation Breeding of Groundnut (Arachis hypogaea L.) 511
16.18 Conclusions and Prospects
One of the impediments in the exploitation of plant genetic resources was transfer-
ring specific genes into ‘ideal desirable genetic backgrounds’(FAO 1998). Convinc-
ingly, a judicious blend of mutation and recombination breeding efforts in groundnut
was successful in bringing mutant genes into ideal genetic backgrounds. Conse-
quently, many distinct groundnut varieties were evolved with improved agronomic
and nutritional traits along with stress tolerance as exemplified by groundnut breed-
ing efforts undertaken in many countries. Besides these, mutation breeding was also
helpful to rectify some of the undesirable features associated with desirable
characters. Many of these mutant varieties have been extensively cultivated by the
farming community and have occupied considerable groundnut area in several
countries. Post-genomic era has come up with ample information on gene families,
copy number and sequence divergence within homologues in cultivated groundnut
genome. Such information will help to design more specific sgRNA construct, which
will selectively mutate the homologue in this polyploid crop for better nutritional
value, abiotic and biotic resistance and allergen- and aflatoxin-free food in
groundnut.

512 A. M. Badigannavar and S. Mondal
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