Rapeseed-mustard is the second most important edible oilseed crop in India after groundnut in terms of area and production. Indian mustard (Brassica juncea L.) is the most commonly cultivated of the oilseed Brassica in India. Globally, India ranks 4th in the production of rapeseed mustard oil. In India, Rajasthan ranks 1st followed by Madhya Pradesh in terms of area and production of rapeseed-mustard. Mustard aphid (Lipaphis erysimi) is the main pest of mustard growing areas, leading to a reduction in yield by 35.4-96.0% and oil content by 5-15%. Aphids can be controlled effectively, economically, and environmentally through the development of an aphid-resistant genotype. The absence of aphid resistance genes in Brassica juncea as well as limited knowledge of trait genetics have hampered aphid resistance breeding in Brassica. Mutation breeding refers to the method of using artificial mutagenesis to obtain new biological cultivars, mainly through chemical or radiation mutagenesis. Induced mutagenesis is a reliable method of introducing aphid resistance within Indian mustard because it has demonstrated its ability to introduce aphid resistance in some crops. Reasons for the delayed development in generating mutant aphid resistant variants of Brassica juncea are the random nature of induced mutagenesis and the lack of particular procedures for screening large numbers of genotypes required in breeding for the selection of tolerant cultivars in mustard. For prospective genotype selection and cultivar development in Brassica juncea, mutant lines having varied aphid resistance along with susceptible and tolerant mustard varieties can be subjected to Genome Wide Association Studies to identify QTLs imparting aphid resistance in mutant mustard.

1. MUTATION BREEDING AS A TOOL
FOR APHID RESISTANCE IN
INDIAN MUSTARD
PBG-591(1+0)
Master’s Seminar
on
Presented by:
Sushrut Mohapatra
2nd year M.Sc.(Ag.)
201222105
1

2. My seminar topic will cover the following
• Introduction
• Area and production of Rapeseed mustard
• Losses caused by aphids
• Symptoms of aphid attack
• Problems faced in aphid resistance breeding
• Mutation breeding for aphid resistance
• Case Study
• Possible mechanism of aphid resistance in mustard mutants
• Commercial mutant varieties released
• Limitations and future prospects
2

3. INTRODUCTION
⁕ Scientific name- Brassica juncea (L.)
⁕ Common names- Chinese mustard, Oriental mustard, Brown mustard,
Rai, Laha
⁕ Native to the sub-Himalayan plains of Northern India
⁕ Allotetraploid (AABB), 2n=4x=36
⁕ Self pollinated
⁕ Oil content- 32-40%
⁕ Brassicas are one of the earliest domesticated crop plants by man
⁕ Rapeseed-mustard is the second most important edible oilseed crop in
India
⁕ 3rd most important source of vegetable oil in world after oil palm and
soybean
⁕ Indian mustard accounts for about 75-80 % of the cultivated area
under rapeseed mustard in the country (https://www.drmr.res.in/)
3
Odisha
o Area- 0.109mha
o Production- 0.048mt
o Productivity- 440kg/ha
o Major growing districts-
Kandhamal, Kalahandi,
Sundargarh
( Source- 5 decades of
Odisha Agriculture
Statistics 2020)

4. 4
Source- World Food and Agriculture – Statistical
Yearbook 2021
Canada- 17%
China-15%
Germany-13%
India-10%
Global trends in rapeseed-mustard area, production
and yield from 2008-09 to 2018-19 (Chauhan et al.,
2020)
Production of rapeseed mustard-
Canada> China> India

5. AREAAND PRODUCTION OF
RAPESEED AND MUSTARD IN INDIA
2.72, 41%
0.77,
12%
0.7,
10%
0.63,
9%
0.59, 9%
1.28, 19%
AREA (Million Hectares)
Total- 6.09mha
Rajasthan Madhya Pradesh Uttar Pradesh
Haryana West Bengal Others
5
Source: Agricultural Statistics at a Glance-2021
4.51, 44%
1.31, 13%
1.28, 13%
0.99, 10%
0.72, 7%
1.3, 13%
PRODUCTION (Million Tonnes)
Total- 10.11mt
Rajasthan
Madhya Pradesh
Haryana
Uttar Pradesh
West Bengal
Others
PRODUCTIVITY- 1511 kg/ha

6. Brassica APHID COMPLEX
Major Aphids
Turnip/mustard aphid [Lipaphis
erysimi (Kalt.)]
Cabbage aphid [Brevicoryne
brassicae (L.)]
Peach potato aphid [Myzus
persicae (Sulzer)]
Petiole gall aphid [Pemphigus
populitransversus (Riley)]
Bean root aphid [Smynthurodes
betae (Westwood)]
6

7. LOSSES CAUSED BY MUSTARD APHID
7
Mustard plants infested by
Lipaphis erysimi
 Lipaphis erysimi is regarded as national pest and one
of the principal restricting factors, producing major
output, yield losses
 India’s average yield of mustard is low in comparison
to other mustard-growing countries chiefly due to
aphid pest causing excessive losses in the yield of the
crop (Raj et al., 2017)
 It decreases yield by 35.4 to 96%, seed weight by
30%, and oil production by 5-15% (Chand et al.,
2022)
 L. erysimi carries 10 non-persistent viruses that
affect plants, such as cabbage black ringspot and
mosaic viruses

8. SYMPTOMS OF APHID ATTACK
 It causes damage to the plants by sucking plant sap from the
tender shoots and flowers of the plant in the beginning and
later sucks the sap from tender pods.
 In heavy infestation, both sides of leaves are infested, the
symptoms of yellowing, curling and then drying of leaves
appear, resulting in development of feeble pods and small
seeds in the pods.
 The excessive excretion of honey dew by the aphid on the
leaves results in the growth of black sooty mould
 On mustard, these aphids prefer flowers to leaves
 The aphid attacks generally during December and continues till
March
8
Infestation of aphids on leaves,
flowers, siliquae

9. CONTROL OF MUSTARD APHID
9
Coccinella septempunctata
Chemical Control
 Spraying Dimethoate 30 EC @2 ml per litre of water
or Thiomethoxam 25 WG @ 0.3 g/l of water
 Imidacloprid 17.8% @ 0.25 ml/l of water
Biological Control
 Release of efficient predator like Lady bird beetle
(Coccinella septempunctata) and parasitoid wasp
(Diaeretiella rapae)
Cultural control
Early sowing before 20th October, Use of aphid resistant
varieties
Genetic control
Incorporation of aphid resistance genes in Indian mustard
through genetic engineering/hybridization
Diaeretiella rapae

10. NEED OF APHID RESISTANT MUSTARD PLANTS
10
Economical
Eco-friendly
Supplements organic farming
Higher yield due to less infestation of aphids
Augmenting varietal resistance can substantially reduce usage of
toxic agrochemicals in crop protection (Dhatwalia et al., 2021)

11. GENETICS OF APHID RESISTANCE
In most cases, aphid resistance is monogenic and inherited as a dominant trait (Smith and
Boyko, 2007)
A single R gene most likely controls the defense of the plant against aphid infection (Liang et
al., 2015)
Resistance also occurs as a result of polygenic quantitative traits.
Several plant resistance genes and their homologs are associated with constitutive expression
of resistance to aphids.
No genes governing mustard aphid resistance have been found till date in Indian mustard
11

12. DEVELOPMENT OF APHID RESISTANT
PLANTS
Hybridisation- Mustard aphid resistance is present in wild
germplasm Rorippa indica. To transfer aphid resistance in
Indian mustard, it needs a bridge species which transfers the gene
to Indian mustard.
Transgenic mustard- Transgenic mustard resistant to aphid has
been developed through stable integration of cea (Colocasia
esculenta tuber agglutinin) gene.
In the plant insect bioassays, the cea expressing Indian mustard
lines exhibited significant enhanced insect mortality.
So far, no aphid-resistant transgenic crop plants have ever been
commercialized due to biosafety and bioethical regulations.
12
Rorippa indica

13. PROBLEMS FACED IN APHID RESISTANCE
BREEDING
13
No source for aphid resistance is presently available in Brassica
juncea (Atri et al., 2012)
Narrow genetic base
Lack of resistant sources within crossable germplasms
Lack of knowledge about trait genetics and specific
procedures for screening large numbers of genotypes
Breeding efforts in Brassica juncea have been largely centred around
high yield and quality traits, and little focus has been given to
maintain a substantial level of insect and/or disease resistance (Kishore
et al., 2022)

14. MUTATION BREEDING FOR APHID
RESISTANCE
14
As a technique, mutation breeding has been widely adopted by plant breeders targeting
pathogen resistance and other abiotic stresses. Few studies, however, have considered
using this approach to develop aphid resistant plant varieties:
Using induced mutagenesis (γ-irradiation) on banana (cv. Lakatan), Cueva et al., (2014)
successfully developed mutants that were repellent and resistant to colonization by banana
aphid
Mutants derived from turnip cultivars were found to be resistant to mustard aphid
(Lipaphis erysimi Kaltenbach) (Zimba et al., 2022)
Using a chemical mutagen, Susrama and Pradnyawathi (2019) succeeded in developing
mutants of common bean, cowpea and yard long bean that showed resistance to the
cowpea aphid

15. MUTATION BREEDING
Mutation breeding refers to the method of using artificial mutagenesis to obtain new
biological cultivars, mainly through chemical or radiation mutagenesis. (Ma et al.,
2021)
Mutation breeding is one of the approaches to enhance spectrum of variability for
characters of agronomic and economic importance
Comparatively, radiation mutagenesis has the characteristics of more complex genetic
mutations and more beneficial mutant phenotypes
Till date 3275 mutants derived from 225 species have been registered in mutant variety
database of IAEA (IAEA, 2019)
15

16. MUTATION INDUCTION 16
Plant Materials: For mutation breeding in mustard, seed is the material
of choice because it is quiescent, easily transported, easy to handle, can
be treated at any time, can be stored, and germinated when required.
Mutagens: Ionizing electromagnetic radiations that include gamma
rays are used and EMS are the most widely used mutagens in
mutation breeding for mustard because of their effectiveness
Induction optimization: Optimum dose of a mutagen may be varied
by varying the intensity or duration of treatment. Optimum EMS
concentration used is 0.5% while for gamma ray, dose is 1000 Gy.
Mutagen treatment: Seeds are exposed to mutagenic doses, they can
be irradiated and planted immediately or presoaked and treated with
chemical mutagen(EMS) or the combination of both methods can be
used, first irradiating using Gamma rays then treating with EMS.

17. 17
Zimba et al., 2022
MUTANT POPULATION DEVELOPMENT
Ideal M2 population size should be
1000-1500

18. SCREENING
Phenotypic screening using aphid index is used for detecting mutants having aphid resistance
Mutant selection through phenotypic screening includes mutation detectable on a single plant basis by
visual inspection combined with appropriate screening procedures for resistance or tolerance to aphids
by observing morphological and physiological traits
These screens can be carried out early in M₂ generations
MOLECULAR METHODS TO SCREEN APHID RESISTANCE
Use of TILLING- TILLING allows for identification of variations in mutant genome providing a
criteria for shortlisting mutants with potential aphid resistance to include in phenotypic screening.
Use of Pathogen Responsive(PR) gene panel (AP2, TIR1, SNAP, MAPK, PRH43 and DREB2B)
developed by Raj et al., (2017) to screen aphid resistance/tolerance in mustard mutants based on
changes in phytohormonal expression pathway
18

19. 19
Ingle et al., 2020

20. CASE STUDY
20
Objective: To induce mutation to study the variability in M2 for qualitative and quantitative
characters and to identify the superior desirable mutants in M2 generation

21. MATERIALS AND METHODS
Dry healthy and genetically
pure seeds of cultivars BIO
902 & Pusa Bold were
divided into six lots of 300
seeds each for giving the
gamma rays treatment
separately and one lot of
BIO902 was kept as control
These 600 seeds of selected
variety were subjected to
irradiation with 900, 1000,
1100, 1200 and 1300 Gy
(Co60 at BARC)
The 300 seeds of each
treatment were treated with
0.5% aqueous solution of
EMS after pre-soaking with
sterilized distilled water for
12 hours
The treated seeds were sown
in the field to raise M1
generation in non replicated
trial
M1 generation was screened
for different morphological
mutants and seeds from each
M1 generation were
harvested separately
M2 generation was raised.
13/12 treatments included
different doses of gamma
rays alone and combination
of gamma rays and EMS
along with untreated check
30 M2 plants were selected
at random per treatment to
record the observations on
yield and yield components
30 observational plants were
scored in field and plants
were rated in 0-9 scale for
powdery mildew infestation
Ten observational plants
were scored in field and
plants were rated in 0-5 for
aphid infestation
21

22. Mutation frequency
Nair et al., 2017

23. RESULTS
23
Nair et al., 2017

24. RESULTS (Contd.)
• Based on yield performance of selected M2 mutants, disease score and aphid index:
 Increase in frequencies over character with increase in doses of gamma rays and EMS in
combination was observed
 12 mutants of BIO 902 and 27 mutants of Pusa Bold having high resistance(<1) to aphid
were identified
 9 mutants of Pusa Bold and 4 mutants of BIO 902 were identified which were highly
resistant/immune to powdery mildew and had high resistance to aphids
 Total 69 M2 plants from different treatments of BIO 902 & 79 mutants of Pusa Bold were
selected mainly on the basis of seed yield, aphid resistance, powdery mildew resistance
24
Performance of untreated check in BIO 902

25. POSSIBLE MECHANISM OF
RESISTANCE IN MUSTARD MUTANTS
• Yadav and Rana, 2018 found high wax content in leaves of RLM-198 and RLM-
514 which resulted in reduced aphid infestation
• Agrani has thick pod coat which makes it tolerant to aphid attacks
• Samal et al., 2021 reported that Kranti (mutant of Varuna) had higher amounts
of antioxidants, tannins, phenols, and ferric ion reducing antioxidant power
(FRAP) which resulted in adverse effects on reproductive period, fecundity and
survival of L. erysimi
25

26. POSSIBLE MECHANISM OF RESISTANCE
IN MUSTARD MUTANTS (Contd.)
Aphid and pathogen resistance genes are often clustered on the same region of the
chromosomes (Zimba et al., 2022). Chromosomal alterations due to induced mutagenesis
are likely to induce genetic variations for both pathogen and aphid resistance traits.
Increased signal transduction of Salicylic Acid/Jasmonic Acid pathway:
Overexpression of large and diverse ensembles of genes in mutants may have increased
signaling of SA/JA pathways which in turn upregulate the genes encoding downstream
defense proteins thus giving rise to resistance to the aphid infestation.
Therefore, it is likely that disease resistant mutants could also resist species of aphid that
are vulnerable to the SA signaling pathway.
Induction of R gene analogue: Mutation may induce resistance gene analogues, as
potential resistance (R)-genes, which have unique roles in the recognition and activation of
aphid resistance responses.
26

27. 27
Source: IAEA Mutant Variety Database
Variety Name Country Name Character Improvement Details Mutant Development Type
Binasarisha-7 Bangladesh Moderately resistant to stem rot,
tolerant to Alternaria blight and
aphid, Bold seed size and higher
number of siliquae.
Direct use of an induced mutant
Binasarisha-8 Bangladesh Moderately resistant to stem rot,
tolerant to Alternaria blight and
aphid
Direct use of an induced mutant
Agrani Bangladesh Early maturity, bigger pod and seed
size, tolerant to Alternaria disease
and aphids, high yield and high oil
content (44%)
Direct use of an induced mutant
RL 1359 India Short duration, high yield, high
TGW, oil content (43%), erect plant
type, tolerant to aphid
Crossing with one mutant
RLM 198 India Tolerant to aphid, Improved oil
content, early maturity (5-6 days)
and high yield
Direct use of an induced mutant
RLM 514 India High oil content, less erucic acid
(11%), early maturity, aphid
tolerant
Direct use of an induced mutant
Safal Bangladesh High seed yield, high oil yield (43-
44%), tolerant to alternaria
disease ,aphids in field conditions
Direct use of an induced mutant
Source: IAEA Mutant Variety Database

28. LIMITATIONS
 Labour intensive screening of large mutant populations to optimise chances of finding desirable
mutations
 Random non-target effects in the genome making it difficult to precisely target specific genes
controlling a desired characteristic
 Treatment of plant material by mutagens invariably kills cells causing a wide range of
deformities and other side effects (sterility) in surviving plants
 Potential mutants usually require several generations of successive propagations or crossing with
other genotypes to exclude undesirable side effects from their genetic background
 Requirement of at least three generation before any stable selection of desired traits in mutants
which leads to 7−9 years of average mutation breeding study
28

29. FUTURE PROSPECTS
29
The improved capability of genomic tools allows for easier identification of
mutants, introgression and molecular characterization of durable resistance to aphid
pests.
Traditional phenotype based mutation breeding can be supplemented with novel
molecular techniques like double haploids, rapid generation cycling, marker
assisted selection, in vitro techniques.
Use of genetic/molecular methods of screening like TILLING, PR gene panel
Using susceptible and tolerant varieties and the mutant population having varied
resistance, Genome Wide Association Studies can be done to identify aphid
resistant genes in the genome and used for further introgression.

30. CONCLUSION
⁂In India, the mustard aphid is the most devastating insect in the Brassicaceae family,
producing major crop losses
⁂ Mutation breeding is still a major method to produce new alleles and new desired traits
within the crop genomes.
⁂Traditional phenotype based mutation breeding can be supplemented with novel molecular
techniques in order to quickly get aphid resistant plants
⁂Gene governing aphid resistance in mutant mustard can be identified and used in
conventional hybridization or through biotechnological tools to transfer aphid resistance in
Indian mustard
⁂Analysis of mutants at molecular level would reinforce the understanding on mechanism of
mutations giving rise to aphid resistance in the era of genomics
30

31. REFERENCES
Atri B, Kumar B, Kumar H, Kumar S, Sharma S and Banga SS. 2012. Development and characterization of Brassica juncea-
fruticulosa introgression lines exhibiting resistance to mustard aphid (Lipaphis erysimi Kalt). BMC Genetics, 13(104): 1-9.
Chand SP, Debnath S, Rahimi M, Ashraf MS, Bhatt P and Rahin SA. 2022. Contextualization of trait nexus and gene action
for quantitative and qualitative characteristics in Indian mustard. Journal of Food Quality, 2022: 1-24.
Chauhan J, Choudhury P, Pal S and Singh K. 2020. Analysis of seed chain and its implication in rapeseed-mustard (Brassica
spp.) production in India. J. Oilseeds Res., 37(2) : 71-84.
Dhatwalia D, Aminedi R, Kalia V, Pandeb V and Bhattacharya R. 2021. Host-mediated attenuation of gut sucrase in mustard
aphid (Lipaphis erysimi) impaired its parthenogenetic reproduction on Indian mustard (Brassica juncea). Pest Manag. Sci.,
78: 803-811.
Ingle AS, Biradar VK, Wawdhane PA, Mahankuda B and Chaple KI. 2020. Screening of mustard mutants for resistance
against mustard aphid, Lipaphis erysimi (Kalt.). Journal of Entomology and Zoology Studies, 8(5): 1779-1781.
Kishore PS, Singh AP, Dewanjee S and Pandey PK. 2022. Mutation breeding as a tool for aphid resistance in Indian mustard
[Brassica juncea (L.) Czern & Coss]. Biological Forum, 14(1): 1111-1118.
Ma L, Kong F, Sun K, Wang T and Guo T. 2021. From Classical Radiation to Modern Radiation: Past, Present, and Future of
Radiation Mutation Breeding. Front. Public Health, 9:1-11.
Nair B, Nartam AT, Badukale MG and Mohurle NA. 2017. Gamma rays and EMS induced mutants in Indian mustard
(Brassica juncea). Journal of Oilseed Brassica, 8(1) : 62-71.
Zimba KJ, Sohati PH, Munyinda K, Roberts JM, Pope TW. 2022. Induced mutagenesis: An underutilised component in the
integrated management of aphid pests in sub-Saharan Africa. Crop Protection, 159:1-6.
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