1) Mutation breeding through chemical and physical mutagens has been employed in Catharanthus roseus to develop mutants with higher alkaloid content and improved agronomic traits. Several mutants with increased root and leaf alkaloids, as well as dwarf and semidwarf mutants, have been isolated. 2) One notable mutant developed was a functional male sterile mutant with indehiscent anthers. Another mutant had a single cotyledon but developed two upon cytokinin treatment, demonstrating cytokinin deficiency. 3) While mutation breeding holds promise for improving this important medicinal plant, more extensive efforts are still needed to develop cultivars with optimized anticancer and antihypertensive alkaloid

1. RANJITHA G.V.
MA1TAG0230

6. • The most popular method employed for creating genetic variability, is
induced mutagenesis through gamma irradiation (Reddy 1977).
• Besides gamma irradiation,chemical mutagens like ethyl-nitroso-urea,
methylnitroso-urea, ethyl-methane-sulphonate (EMS) and sodium azide
(SA) are also used for mutation-assisted breeding.
• Among all these SA is considered as safe and has capability of producing
high frequency of mutation.

7. Materials and Method
• Plant material : TFDRG 5, a recombinant line from the cross between
VG 9514 (Varman 1999) and TAG 24 (Patil et al, 1995) has a red seed
coat, highly resistant to rust and moderately to late leaf spot
(Badigannavar et al., 2005). VG 9514 and TAG 24 have red and rose
testa respectively.
• Mutagenesis : Seeds of TFDRG 5 (200 each) treated with gamma rays
(200 & 300 Gy) or sodium azide (NaN3 1, 2 & 3 mM) or combination
of both. For NaN3 treatment, seeds were initially soaked in distilled
water for 4 h and then treated with NaN3 at 1mM, 2mM and 3 mM
for 17 h at pH 7.0.

8. • A total of 215 variants were isolated from an M2 population
consisting of 20,619 plants. Out of 11 different treatment, the
combination of ‘200 Gy + 3 mM NaN3 ’ was ideal with the highest
mutation frequency (2.48%) in the M2(Table 1).

10. • Among 215 variants in the M2 , 63 true breeding mutants for plant
height, plant habit, leaf related traits, flower colour, pod-traits, seed
size and testa colour were identified in M3 .
• Maximum numbers of mutants were obtained for the leaf related
traits. Among leaf related mutants, mutant phenotype like light green,
small leaf, narrow leaf, disease mimic leaf, mosaic leaf, virescent leaf,
twisted leaf and Fused Terminal Leaflet (FTL) were isolated.
• Among plant height mutants seven dwarf mutants were isolated (Fig.
1). Besides, ten rose seed coat colour mutant, 21 large pod mutant,
one for hard kernel and two mutants for plant habit were identified in
the M2.

11. • VB mutants arose due to reversion as one of the progenitors of the
TFDRG 5 is VG 9514, which is a Virginia bunch, red seed coat and
immune to both rust and Late Leaf Spot (LLS) (Varman 1999).
Between these two VB mutants M 95 was susceptible to rust but
resistant to LLS whereas M 101 was resistant to both the diseases.
• Thus it showed, that even in the mutation for a specific trait (like VB
plant type), the mutants were diverse in respect of other characters.

12. The Fused Top Leaflet (FTL) mutants
(Fig. 2) in our study, produced three
leaflets and the top leaflet is a result of
fusion between top two leaflets in the
parent.
Besides, the rachis between two pairs
of leaflets is completely absent in these
FTL mutants.
Thus, the whole leaf appears as a
trifoliate. In this connection the mutant
can be considered as a missing link/
transition state between compound
pinnate leaves and compound foliate
leaves.
These new FTL mutants with their
fused top leaflets are novel in nature
because they show 100% expressivity
between the plants and the rachis
between the pair of leaflets is
completely absent.

16. • In 21 large seed mutants (Fig. 5), seed size increased by 15 to 53% as
compared to the parent.
• Of these, 18 are with red testa like parent and 3 are with rose testa.
Three of these large seed mutants showed superior pod and seed
weights by 15.7 to 46.3% in comparison to parent (Table 3) at M4 .
• Among these three, two mutants namely M59 and M78 showed
consistent pod yield performances at both M3 and M4 .
• Variability among the mutants was also observed in terms of their
disease resistance and the agronomic characteristic like Hundred
Kernel Weight (HKW)

18. • Ten rose testa mutants were identified in the M2. M3 segregation
gave 3 plants with rose testa and 1 with red (Fig. 6). Further,
M4segregation into 1 (all rose):2 (3 rose: 1 red): 1 (all red) confirmed
single gene dominant mutation for rose testa.

20. Review Paper on Mutation Breeding as Applied in
Groundnut (Arachis Hypogea L.) Improvement
• As a legume, groundnut improves soil by fixing nitrogen biologically
without consuming non-renewable energies and without disturbing
agro-ecological balance.
• Aflatoxin is one of the major problems in groundnut, which hinders
not only the domestic consumption but also export of groundnut
since the international regulation for minimum standards for
aflatoxin contamination is becoming stringent.
• To reduce the harmful effect of Aflatoxin and meeting other
agronomic and breeding objectives, mutation breeding is an
important breeding strategy.

21. • Mutagenesis can be physical (the use of physical agents) or chemical
(the use of
• chemical agents) to create variability. Development of high-yielding
peanut mutants through chemical mutagenesis such as
• Flower injection of ethyl methane sulfonate (EMS), Seed treatment
with chemical mutagens such as sodium azide (NaN3) was resulted in
yield increment over untreated local varieties.
• Physical mutagens include electromagnetic radiation, such as gamma
rays, X-rays and UV light and particle radiation such as fast and
thermal neutrons, ß and alfa particles were also used in ground nut
improvement.

22. • Development of high-yielding peanut mutants through chemical
mutagenesis
1. Flower injection of ethyl methane sulfonate (EMS)
• injection of 0.3% EMS into flowers of Huayu 16 it was able to develop a
high-yielding peanut
• The improved variety, Huayu 40 has an erect growth habit and sequential
branching pattern.
• As compared with its wild type (Huayu 16), Huayu 40 possesses faster
growing and darker green foliage
• In addition, also reported that leaf water content, chlorophyll a and b
content of Huayu 40 were significantly higher than those of Huayu 16.

23. Breeding for Aflatoxin Resistance
• Aflatoxins are the secondary metabolites produced by Aspergillus species in
several agricultural products.
• These aflatoxins are found to have carcinogenic, teratogenic and hepatotoxic
effects in the consumers . Of various agricultural commodities, groundnut is a
major oilseed crop that is often contaminated by these aflatoxins .
• Aflatoxin is one of the major problems in groundnut, which hinders not only the
domestic consumption but also export of groundnut
• since the international regulation for minimum standards for
aflatoxin contamination is becoming stringent.

24. • Aflatoxin contamination is a qualitative problem induced by
Aspergillus flavus and A. parasiticus.
• Aflatoxins are stable small molecules and cannot be destroyed by
heat treatment or during processing.
• As reported by some authors, approximately 80% reduction in total
aflatoxin content over the control used after treatment with Ajowan
seed extracts .

28. • High yield, branching habit, non-shattering and high oil within the
local populations, a number of varieties like HC.1, HC.2, HC.3, HC.4,
HC.5, HC.6, HC.7, HC.8 were isolated in Andhra Pradesh. Out of these,
HC.6 and HC.8 became popular among the farmers.
• However, the yield improvement was only 10 to 20% over local
varieties. Then came the breakthrough in varietal front in late sixties
with the isolation of a short duration mutant named as 'Aruna' while
releasing for cultivation from the thermal neutron treatment of HC.6.

29. • Until the advent of this mutant, all improved strains of castor in India
were of long duration taking 220-280 days for maturity.
• This is the first short duration variety maturing within 120-150 days
developed in castor.
• The distinguishing characteristics of Aruna and the parent are given in
Table.

31. • The alteration of several morphological features of HC.6 through
mutation breeding facilitated Aruna variety to be more responsive to
intensive crop management practices.
• It became popular all over the country particularly Andhra Pradesh
and Karnataka. Subsequently, another short duration variety, Bhagya
was developed from a cross between High oil line (HO) and M..415
which was released for cultivation in 1974.
• Apart from broad genetic base ensuring population buffering, Bhagya
is earlier and shorter than Aruna.
• Simultaneously another variety, Sowbhagya was developed from a
double cross involving Aruna

32. • Sowbhagya is an entirely different variety in respect of plant type
and growth duration.
• The converging dwarf plant type and slightly longer duration of the
variety made it more suitable for intercropping systems.
• Later, a number of varieties have been developed and released for
cultivation from different centres all over India .

33. Catharanthus roseus (L.)
Chromosome no : 2n=16
Common name : periwinkle
Family : (Apocynaceae),
Origin : native of south eastern and eastern Madagascar,
• It has been popularly known for its considerable medicinal value. Its
medicinal values were known even in 50 BC.
• It possesses largest number of alkaloids in plant kingdom. More than
125 alkaloids of the indole and dihydroindole groups have been
isolated and characterized from different organs.

34. Mutation breeding in Catharanthus roseus (L.)
Overview
• Induced mutagenesis is best approach for creation of genetic
variability but most of interest is paid towards crop plants with only a
few exceptions. But even widely used medicinal plants could receive a
little attention in this direction.
• Catharanthus roseus is an important plant medicinal plant yielding
antihypertensive alkaloid ajmalcine and anticancerous alkaloid
vincristine and vinblastine. Despite of considerable work had been
done on biotechnological aspect of C. roseus alkaloid production but
relatively little efforts has been made for its improvement by
mutation breeding approach.

35. • The medicinal importance of this plant has increased considerably
because of the discovery of six anti-cancerous activity containing
alkaloids.
• Among them only vincristine and vinblastine are active in
humensystem.
• The credit of discovery of these two alkaloids goes to Nobel et al.
• C. roseus also owns its importance due to the presence of
antihypertensive alkaloids such as ajmalcine and serpentine in roots
The annual world demand for vincristine, vinblastine and ajamalcine is
very high.

36. • Mutation Breeding is a supplementary approach for creation of
genetic variability, which is also applied in improvement of C. roseus
(L.) G. Don at limited extent
• Vekaateswarlu et al. Bhattacharjee, Chatacharjee et al. Attempted
induced mutagenesis in C. roseus for upgradation of leaf alkaloid
content and isolated several morpho- chemotypes.

37. • Kulakarni et al reported three mutants, one dwarf, one semidwarf
with high alkaloid content in the roots and one mutant with wavy leaf
margin and high alkaloid content in its leaves, were isolated after
chemical mutagenesis with EMS and N-nitroso-N etylurea in the variety
‘Nirmal’.
On the basis of inheritance studies on morphological traits, they found
that the mode of inheritance of all three mutants was controlled by
monogenic recessive genes.

38. • Kulakarni et al reported a mutant with indehiscent anther (functional
male sterility),
• following an induced mutagenesis programme, which was taken up
with an objective of inducing mutants with high alkaloid content in
variety ‘Nirmal’. The mutant had smaller anther, with lesser number
of pollen grains.
• Rai et al reported EMS induced mutant in which embryo had only
one cotyledon but upon supplementation with the natural cytokinine,
kinetine it developed two cotyledons.
• This is perhaps the first plant mutant in which development stage
specific cytokinin deficiency has been demonstrated

39. • Kulkarni et al. reported a periwinkle variety‘Dhawal’ a high alkaloid
producing variety produced by chemical mutagen treatment of seeds
followed by rigorous selection in widely cultivated variety ‘Nirmal’.
• Anjalica et al.carried out a mutagenesis experiment using gamma rays
and EMS as mutagens and reported six morphological mutants viz.
very tall (vt), dwarf with obvate leaves (dwob), medium tall with small
leaf area (mtsl), non trichomous (nt), upright oriented elliptical leaf
(Upef) and spoon shaped leaf (Spl) and described dwob mutant as
most promising mutant for cultivation in India.

40. • Baskaran et al reported two EMS induced macro-mutants (‘necrotic
leaf’ and ‘nerium leaf) of periwinkle with enhanced contents of total
root and leaf alkaloids and anticancer leaf alkaloids, vincristine and
vinblastine than the parental variety. Their leaf and root yields were,
however, significantly lower than their parental variety.

41. • It Evident form this review, although induced mutagenesis is an
efficient approach for the development of improved genotypes but it
is not attempted at adequate level for the improvement of C. roseus.
• Keeping this in view more concerted efforts on this direction are
needed which may leads to development of high anticancerous and
antihypertensive containing ‘ideochemocultivars’ which helps in
decreasing extraction cost as well as market value of these alkaloids.