spectrum and frequency of macromutations induced in

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Turk J Biol35 (2011) 221-231 © TÜBİTAKdoi:10.3906/biy-0902-20

Spectrum and frequency of macromutationsinduced in chickpea (Cicer arietinum L.)

Aijaz A. WANICytogenetics and Reproductive Biology Laboratory, Department of Botany,

University of Kashmir, 190006, Srinagar J&K - INDIA

Received: 18.02.2009

Abstract: The present investigation was undertaken to study the frequency and spectrum of morphological mutantsinduced by gamma rays and EMS in chickpea (Cicer arietinum L.). The seeds of 2 varieties of chickpea, Pusa-212 andPusa-372, were treated with gamma rays (150 Gy, 200 Gy, 300 Gy, and 400 Gy), EMS (0.1%, 0.2%, 0.3%, and 0.4%) andcombination treatments (200Gy+0.2%EMS, 300Gy+0.2%EMS, 200Gy+0.3%EMS, and 300 Gy+0.3%EMS). The M2population was carefully screened for various viable macromutations. A wide spectrum of viable morphological mutationsaffecting almost all parts of the plant were isolated in the M2 generation. The most striking mutants isolated included tall,dwarf, broad-leaved, white-flowered, bold-seeded and high-yielding mutants. Differences in varietal response to differentmutagens was clearly evident as both of the varieties differed in the spectrum and frequency of mutations induced.Combination treatments were most effective in inducing a wider spectrum and maximum frequency of macro mutations,followed by EMS. Of all the morphological mutations, the frequency of leaf mutations was maximum, followed by plantheight and seed mutants. Most of the macro mutants were confirmed to be true-breeding in the M3 generation except forthe highly sterile ones. Some mutants were specifically isolated in a particular mutagen type. The results indicate thatinduced mutability is governed by the genotype of the material used and that the mutagenic specificity for different traitsvaries.

Key words: Cicer arietinum L, gamma rays, EMS, macromutations

Nohutta (Cicer arietinum L.) indüklenen makro mutasyonlarınfrekansı ve spektrumu

Özet: Bu araştırma nohutta EMS ve Gama ışınları ile indüklenen morfolojik mutatların spektrumu ve frekansınıaraştırmayı üstlenmiştir. Nohuda ait iki varyetenin tohumları, yani Pusa–212 ve Pusa–372, gamma ışınları (150 Gy, 200Gy, 300 Gy ve 400 Gy), EMS (0,1%, 0,2%, 0,3% ve 0,4%) ve her ikisinin birleşimi (200 Gy+0,2% EMS, 300Gy+0,2% EMS,200 Gy+0,3% EMS ve 300 Gy+0,3% EMS) ile muamele edilmiştir. M2 popülasyonu görülebilir çeşitli makro mutasyonlariçin dikkatlice taranmıştır. Bitkinin hemen hemen tüm kısımlarını etkileyen, görülebilir morfolojik mutasyonların genişbir spektrumu M2 soyunda izole edilmiştir. İzole edilen en çarpıcı mutatlar arasında, koyu tohumlu, beyaz çiçekli, genişyapraklı, bodur, uzun ve yüksek verimli mutatları vardı. Farklı mutajenlere çeşitli cevaplardaki farklılıklar belirgindi; heriki çeşit, indüklenen mutasyonların spektrumu ve frekansı açısından farklılık göstermiştir. Makro mutasyonlarınmaksimum frekansı ve daha geniş spektrumuna sebep olmada en etkili yöntem kombine edilen işlemlerdi, en etkili ikinciyöntem ise EMS idi. Bütün morfolojik mutasyonlar arasında frekansı en yüksek olanı yaprak mutasyonlarıydı, ondansonra tohum mutatları ve bitki boyu gelmiştir. Makro mutatların çoğu yüksek seviyede verimsiz olanlar hariç M3 soyunda

IntroductionChickpea (Cicer arietinum L.) is the third most

important food legume in the world, grown in 40countries over an area of about 11.2 million hectares,with a production of 9.2 million tons and an averageyield of 818 kg/ha (1). Currently, the productivity ofchickpea is very low and has remained stagnant inrecent years, probably due to limited genetic variationavailable in the crop (2). Moreover, the conventionalapproaches of plant breeding have exploited theavailable genetic variability in chickpea, which has inturn led to a narrow genetic base in this crop.Mutagen-induced quantitative variation not onlyserves as an alternative source of germplasm fornatural variation, it is also useful in generatingappropriate linked gene complexes which areresponsible for improvement in yield and othercharacters of economic interest. It has beendemonstrated by several researchers that geneticvariability for several desirable characters can beinduced successfully through mutations, and itspractical value in plant improvement programmes hasbeen well established (3-5). Many physical andchemical mutagens have been used for induction ofuseful mutations in a number of crops (6-15). AtFAO/IAEA, an official mutant database has beenmaintained since 1966 (16,17), and since then morethan 2300 officially released mutant varieties in 59countries have been listed (18). The main advantageof mutation breeding is the potential to improve 1 or2 characters without changing the rest of thegenotype. In several mutation-derived varieties, thechanged traits have resulted in synergistic effects onincreasing the yield of the crop, improving agronomicinputs, crop rotation, and consumer acceptance (5).

Macromutations (distinct qualitative geneticvariations) are easily identified and therefore can beefficiently selected for varietal development programs,or otherwise exploited through cross breeding (19).Chickpea offers greater opportunities for the isolation

of useful mutations. Induced mutagenesis is thereforeof special significance, and can be exploited to isolatepromising mutants of economic as well as academicinterest. The success of this technique in cropimprovement has been amply demonstrated over theyears across plant species, including economicallyimportant crops (16,20).

In view of the above considerations, an attempt hasbeen made to induce alterations in the genotype toenhance genetic variability so as to isolate somepromising macro mutations that could lead toimprovement in yield and other economicallyimportant traits through the use of physical andchemical mutagens in chickpea.

Materials and methodsThe certified seeds of 2 varieties of chickpea (Cicer

arietinum L.), var. Pusa-212 and var. Pusa-372, wereprocured from the Genetics Division, IARI, NewDelhi. Dry and healthy seeds (moisture content =10%-12%) of both varieties were treated with differentdoses/concentrations of gamma rays, EMS and theircombination treatments (21). The M2 populationconsisted of 1500 seeds in each treatment with 500seeds in each replication plot. The M2 population wascarefully screened for various viable macromutationsfrom the date of emergence until the attainment ofcrop maturity. Various mutants were identified andtagged for separate harvesting. The frequency ofmorphological mutations was calculated on the basisof total number of M2 plants in the respectivemutagen. The frequency of each mutant type was alsocalculated on the basis of the total number of M2plants screened in all the treatments in a particularvariety. Finally, the overall frequency ofmacromutations isolated in each mutagen (gammarays, EMS and combination treatments) wascalculated on the basis of the total number of M2plants screened in a variety. The viablemacromutations were carefully analyzed for pollen

Spectrum and frequency of macromutations induced in chickpea (Cicer arietinum L.)

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hatasız üretim doğrulandı. Bazı mutatlar özel bir mutajen tipte spesifik bir biçimde izole edildi. Sonuçlar, indüklenenmutabilitenin kullanılan materyalin genotipine bağlı olduğuna ve mutajenik özgüllüğün farklı özelliklere göre değiştiğineişaret etmiştir.

Anahtar sözcükler: Cicer arietinum L, gama ışınlar, EMS, makro mutasyonlar

fertility and seed set. Pollen fertility was computedusing a simple acetocarmine test. The purpose was toavoid mutants with gross chromosomal aberrationsand high sterility and to select mutants with highfertility and less chromosomal aberrations for futuregenerations. Seeds of fertile macromutations wereused to raise the M3 generation to determine theheritable nature of the mutant trait.

Results and discussionA wide range of viable morphological mutations

affecting almost all parts of the plant and seedcharacteristics were isolated in the M2 generation oftreated populations (Tables 1-2, Figures 1-20). Nomutant plant was isolated in the control. All thesemacromutations were classified on the basis of trait

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Table 1. Frequency of morphological mutations induced by gamma rays, EMS and their combination treatments in Cicer arietinum Var.Pusa -212 and Var. Pusa -372

Gamma rays EMS Gamma rays + EMS Total2

Var. Var. Var. Var. Var. Var. Var. Var.Mutant type Pusa -212 Pusa - 372 Pusa - 212 Pusa - 372 Pusa - 212 Pusa - 372 Pusa - 212 Pusa - 372

(4937) (4961) (4532) (4762) (4621) (4525) (14090) (14248)

Plant HeightTall 03 - 05 04 08 06 16 10Dwarf 05 05 08 05 12 08 25 18

Growth HabitBushy, compact and dark green - 03 07 06 09 09 16 18Prostrate 04 01 - - 03 04 07 05

LeafBroad-leaved, gigas 06 04 - - 04 03 10 07Small, narrow-leaved 02 07 06 05 07 08 15 20Trifoliate type - - - - 06 - 06 -Reduced number of leaflets - - 03 - 07 04 10 04Deep serrated leaflets - 03 04 05 02 - 06 08

FlowerDouble flower - - 03 - 05 03 08 03Violet /white flower - - - 05 - - - 05Non-flowering, vegetative 03 06 05 08 08 12 16 26

MaturityEarly flowering 05 - 04 03 - 02 09 05Late flowering 02 02 05 04 05 03 12 09

Pod/SeedBold 06 04 - - 03 05 09 09Small 02 02 05 06 06 04 13 12

YieldBranches per plant 02 03 05 05 07 05 14 13Pods per plant 05 06 07 04 06 06 18 16

Total1 45 46 67 60 98 82 2103 1883

1. Overall frequency of all mutant types in each mutagen, 2. Overall frequency of each mutant type in all 3 mutagens, 3. Overall frequencyof mutants isolated in each variety Note: Figures in parentheses indicate total number of M2 plants screened.

affected and their frequency was computed as in thecase of chlorophyll mutations (22). There weredifferences in the mutation spectrum both betweenthe varieties and among the mutagens. However,many similarities were noticed between the 2 varietieswith respect to the spectrum and frequency of aparticular mutation. The macromutations induced inthe present study are described below.

Plant height mutantsThe plant height in the control populations ranged

from 50-65 cm. Tall mutants with a remarkableincrease in plant height (70-85 cm) were isolated inall mutagens except gamma rays in var. Pusa-372.Combination treatments (200Gy+0.2%EMS &300Gy+ 0.2%EMS) induced the maximum frequency

of tall mutants. The mutants produced fewerbranches, and the pod number was also low ascompared to the control. The frequency of tallmutants was 0.11% (var. Pusa-212) and 0.07% (var.Pusa-372) of the total M2 plants screened (Table 2).

Dwarf mutants were isolated in higher-dosetreatments of all the mutagens. The plant height ofdwarf mutants ranged from 20-30 cm with profusebranching at the base, short internodes and smallerpods. The proportion of dwarf mutants was 0.18%(var. Pusa-212) and 0.13% (var. Pusa-372) of the totalM2 plants. However, their frequency was maximumin combination treatments (200Gy+0.3% & 300Gy+0.3 % EMS). Both tall and dwarf mutants werefound to be true-breeding in the M3 generation.


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Table 2. Proportion* of different morphological mutations isolated in M2 generation in chickpea (Cicerarietinum L.).

Mutant Type Var. Pusa-212 Var. Pusa-372

Tall 0.11 0.07

Dwarf 0.18 0.13

Bushy, compact, dark green 0.11 0.13

Prostrate 0.05 0.03

Broad-leaved, gigas 0.07 0.05

Small, narrow-leaved 0.11 0.14

Trifoliate type 0.04 -

Reduced number of leaflets 0.07 0.03

Deep serrated leaflets 0.04 0.06

Double flower 0.06 0.02

Violet/White flower - 0.03

Non-flowering, vegetative 0.11 0.18

Early flowering 0.06 0.03

Late flowering 0.08 0.06

Bold pod/seed 0.06 0.06

Small pod/seed 0.09 0.08

Branches per plant 0.1 0.09

Pods per plant 0.13 0.11

* Frequency (%) based on total number of M2 plants screened.

Growth habit mutantsSome bushy, compact and prostrate mutants were

isolated in this category. The bushy mutants weredwarf with condensed internodes, compactness ofbranches and closely placed leaflets on the rachis.Pollen sterility was very high (60%-80%), whichultimately resulted in the failure of pod formation.The prostrate mutants, on the other hand, showed atrailing tendency. Pollen sterility ranged from 35% to50% and seed set was very low. These mutants wereisolated from 400Gy gamma rays and300Gy+0.3%EMS treatments only. The frequency ofbushy mutants was 0.11% and 0.13% and that ofprostrate mutants was 0.05% and 0.03% of the totalM2 plants screened in var. Pusa-212 and var. Pusa-372,respectively (Table 2).

Mutants affecting leaf morphologyOne of the most common alterations due to

mutagenic treatments was modification of leaf shape.Different types of leaf shape mutants isolated in M2generation included broad-leaved, narrow-leaved,trifoliate, reduced number of leaflets and deepserrated leaflets (Figures 1-9). The frequency of thesemutant types out of the total M2 plants is shown inTable 2. All these mutant types were more frequentlyinduced by combination treatments (200Gy+0.3% &300 Gy+0.3 % EMS). Gamma rays (300 Gy) and EMS(0.2% & 0.3%) induced a few types of such mutants(Table 1).

The broad-leaved, gigas mutants isolated fromgamma rays and combination treatments were tallwith a lower number of primary branches. Secondarybranching was profuse, and leaflets were broader butfew pods were produced. In contrast, the narrow-leaved mutants had small or narrow needle-likeleaflets. Branching was normal, but flowering andmaturity was delayed with few pods produced atmaturity. Pollen sterility in these mutants ranged from60% to 80%. One characteristic leaf mutant wasisolated as an apinnate mutant in the higher dosecombination treatment (300Gy+0.3% EMS) in var.Pusa-212.This mutant showed profuse branching, andthe leaves were highly reduced, with only three leaflets(instead of 12-14 in the control) on a small petiole-like structure. Since the leaf rachis was absent, thismutant was named an apinnate mutant. The floralparts of the mutant were poorly developed and

rudimentary. No seed set was recorded in this mutant.Almost all types of leaf mutants (except apinnate andnarrow-leaved) were found to be true-breeding in theM3 generation.

Flower mutantsMutants isolated in this category included double-

flower, white-flower and non-flowering vegetativemutants. Double-flower mutants (Figure 8) wereisolated in 0.2%EMS and 200Gy+0.2%EMStreatments only. Plants were tall with broader, darkgreen and deeply serrated leaflets. Each peduncleconsisted of two normal flowers in which only one ofthe flowers developed into a pod. Pollen sterilityranged from 40% to 50% and very few bold seeds wereproduced. The frequency of these mutants was 0.06%(var. Pusa-212) and 0.02% (var. Pusa-372). One true-breeding mutant was isolated from 0.3% EMS in var.Pusa-372. This mutant was characterized by violetflowers which gradually turned white instead of pinkflowers in the parent variety (Figure 13). Seed set inthis mutant was normal.

In addition to the above, some non-floweringmutants with no seed set (0.11% in var. Pusa-212 and0.18% in var. Pusa-372) were isolated from higherdose treatments of all mutagens (Table 2)

Mutations affecting maturitySome early flowering mutants that flowered 7-10

days earlier and late flowering mutants whereflowering was delayed by 10-15 days as compared tothe control population were isolated in themutagenized population. These mutants showed25%-35% pollen sterility and were found to be true-breeding to their types in M3.

Pod and seed mutantsIsolated from gamma rays (300Gy & 400Gy) and

combination treatments (200Gy+ 0.2%EMS &300Gy+0.2% EMS) only, the frequency of bold seededmutants was 0.06% of the total M2 plants in bothvarieties. These mutants were associated withvigorous growth, broad leaflets, large-sized flowersand increased pod size with 1 or 2 bold seeds in eachpod. However, the degree of boldness varied indifferent mutants. Three bold-seeded mutants, whichwere designated as Pusa-212-A, Pusa-212C and Pusa-212F, were quite distinct from the rest of the bold-seeded mutants and were also isolated as

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high-yielding true-breeding mutants (23). Somemutants with very small sized seeds associated withdwarfness and compactness were also isolated in bothvarieties.

High yielding mutantsOne category of such mutants showed an

increased number of primary branches [10-13] or

secondary branches [30-40]. The mutants hadluxuriant growth and were associated with increasednumber of flowers and consequently more pods perplant [170-220] as compared to 128-162 pods perplant in the control. The frequency of such highyielding mutants was 0.10% (var. Pusa-212) and0.09% (var. Pusa-372). Another category of these


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1 2b

65

4

3

Figures 1-6. Different types of leaf mutants isolated in treated populations of chickpea(controls on left side) 1. Mutant with broad leaflets and stipules. 2 a, b.Two broad-leaved mutants showing deep serrations on the margin. 3.Mutant with reduced intermodal length and small narrow leaflets. 4.Broad-leaved mutant with reduced number of leaflets. 5. Bushy compactmutant with close leaflets. 6. Mutant with long rachis and increasednumber of leaflets.

2a

mutants showed an increased number of pods only.These plants were associated with tallness ordwarfness with almost normal branching, butproduced an increased number of pods [190-240].Seeds were smaller than the parental variety. The

frequency of these true-breeding mutants was 0.13%and 0.11% in var. Pusa-212 and var. Pusa-372respectively (Table 2). Both these mutant types wereisolated in the lower or intermediate mutagenictreatments.

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7 8

9 10

Figures 7-10. Various branching mutants isolated in treated populations of chickpea. 7.Control branch showing arrangement of leaflets. 8. Double flower mutantwith reduced rachis and number of leaflets. 9. Axillary branch mutantshowing small branches arising from leaf axils. 10. Mutant showingincreased number of primary branches.

In general, combination treatments induced notonly maximum frequency but also more types ofmorphological mutations. EMS in turn was moreefficient than gamma rays except for prostrate, broad-leaved and bold-seeded mutants, which were absentin EMS treatments (Table 1). Of the totalmorphological mutations, the frequency of leafmutations was highest, 0.33% and 0.28% in var. Pusa-212 and var. Pusa-372 respectively (Table 2). Amongthe three mutagens, combination treatments inducedthe highest frequency (2.12%) in var. Pusa-212 and(1.81%) in var. Pusa-372 (Table 3). EMS was almostequally efficient in both varieties, whereas gamma

rays proved to be more efficient in var. Pusa-372. Thevarietal response to different mutagens is also clearlyevident from Tables 1-2. Most of these morphologicalmutants have also been reported previously inchickpea (8,24), sesame (25), mungbeen (13,26),urdbean (7), lentil (10,27), Vicia faba (11) etc. Variousinvestigations suggest that the possible cause of thesemacromutations may be chromosomal aberrations,small deficiencies and duplications and most probablygene mutations (28). Several researchers havereported that these viable mutations are monogenicand recessive in nature, controlled by one or morerecessive genes (29,30). In addition to the simple


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11 12

13 14

Figures 11-13. Flower mutants isolated in treated populations of chickpea. 11. Controlplant with pink flowers. 12. Non-flowering vegetative mutant with broadleaflets and reduced rachis. 13-14. White flower mutant and its pods.

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Figures 15-20. Plant height mutants isolated in treated populations of chickpea. 15-16.A dwarf mutant with its pods. 17-18. Semi-dwarf mutant and its pods.19-20. Mutant showing trailing growth habit and its pods.

15 16

17 18

19 20

Table 3. Frequency* of morphological mutations obtained with different mutagens in M2 generation in chickpea(Cicer arietinum L.).

Percent mutated plants

Variety Gamma rays EMS Gamma rays + EMS

Pusa-212 1.32 1.48 2.12

Pusa-372 0.93 1.26 1.81

* Frequency (%) based on total morphological mutations screened in each mutagen type.

viable mutations, multiple mutations affecting morethan one character, particularly those of leaf and seedmutants found in the present study, have beenreported to be due to mutation of the pleiotropic gene,mutation of gene clusters, and loss or rearrangementof chromosomal segments (31,32). Gamma rays areknown to influence plant growth and development byinducing cytological, biochemical, physiological, andmorphogenetic changes in cells and tissues (33). EMStreatments mainly cause point mutations, but smalldeletions and other chromosomal rearrangementsalso occur (34,35). The morphological mutantsdepicted in Table 1 were proved to be true-breeding inthe M3 generation, except some highly sterile ones.The more drastic effects of gamma rays and EMS onthe treated populations were eliminated through anefficient selection procedure in the M1 generation,where only normal looking plants were selected andthe plants with gross morphological changes, highchromosomal aberrations and more than 50% sterilitywere rejected in each treatment. In this context, it maybe concluded that the mutations isolated in thepresent study might have been due to small deletionsor point mutations. However, whether the mutants

affected by multiple traits were due to mutations insingle genes with pleiotrophic effect or multiple genesacting as one functional gene complex is not clear.One possible way to clarify this is to backcross thesemutants with their respective parents and determinethe segregation pattern of the traits involved. Thedifferences in varietal response to mutagenictreatments observed in the present study have alsobeen confirmed by the abovementioned researchersin different crops. In conclusion, the morphologicalmutants isolated in the present study includedmutants with agronomically desirable features whichcould possibly be utilized in future breedingprograms.

Corresponding author:Aijaz A. WANICytogenetics and Reproductive Biology, Department of Botany, University of Kashmir,190 006 Srinagar, J&K - INDIAE-mail: [email protected]


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