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Research Article
 

Selection for Novel Mutations Induced by Gamma Irradiation in Cowpea [Vigna unguiculata (L.) Walp.]



Festus Olakunle Olasupo, Christopher Olumuyiwa Ilori, Brian Peter Forster and Souleymane Bado
 
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ABSTRACT

Background and Objective: Induced mutation is a valuable tool used by plant breeders for creating new variation where crop genetic diversity is insufficient. The study was conducted to select mutants that possess novel morpho-agronomic traits from M2 generation. Materials and Methods: Seeds obtained from M1 plants derived from gamma irradiated seeds of eight cowpea accessions were advanced to M2 generation. The M2 plants were screened and scored for mutant phenotypes on the field. Selected mutants were advanced to M3 and M4 generations to confirm their mutant phenotypes and genetic stability. Results: Observed mutation spectra and frequencies varied across all the accessions and radiation treatments. New cowpea mutants with novel phenotypic and agronomic traits were selected from five out of the eight accessions studied. The frequencies of yellow and white seedling (albino) mutants were higher than other mutants in all cowpea accessions and across radiation treatments. Mutation rates were higher (0.0057<1.745) in the four elite cultivars, whereas, lower range of mutation frequencies (0<0.4013) were recorded in Ife brown (IB) and its derivatives. The tall-erect non-branching trait of the mutant IB-ER could be introgressed to develop tall and erect varieties that may be useful for mechanized cowpea production. Conclusion: Mutants selected in this study could be of benefit for cowpea improvement, genetic analysis, biochemical and physiological studies.

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Festus Olakunle Olasupo, Christopher Olumuyiwa Ilori, Brian Peter Forster and Souleymane Bado, 2018. Selection for Novel Mutations Induced by Gamma Irradiation in Cowpea [Vigna unguiculata (L.) Walp.]. International Journal of Plant Breeding and Genetics, 12: 1-12.

DOI: 10.3923/ijpbg.2018.1.12

URL: https://scialert.net/abstract/?doi=ijpbg.2018.1.12
 
Received: December 05, 2017; Accepted: April 20, 2018; Published: June 04, 2018



INTRODUCTION

Breeding for sustainable crop improvement depends to a large extent on the availability and accessibility of intra-specific genetic diversity of species. Genetic variation provides the resources for adaptation under artificial or natural selection and ultimately this has its origin in mutation1. The history of agriculture revealed that spontaneous mutant traits have enabled the domestication of many plant species with great benefits to man. Mutation has been the main driver for evolution and hence speciation and domestication of both crops and animals2. The germplasm of naturally occurring genetic variability of crop species are kept and maintained by plant breeders because of their rarity, efforts are still being made to create novel variability through induced mutations by physical or chemical agents3 for the purpose of crop improvement. Plant breeding has gone through many developmental stages which include discovery and utilization of various induced mutation techniques for crop improvement4. Therefore, mastering of mutation breeding techniques may become a crucial complementary to further success in the breeding of many crop species. Furthermore, there are three reasons for which plant breeders should pay more attention to mutation induction for breeding programmes. First, for some crops, especially the cereals, the breeding intensity has been so great that for some ecological regions it will be increasingly difficult to achieve further progress only from germplasm already existing and readily available. The second reason is the rapid and alarming erosion of humans genetic resources. These resources are vital if sustained progress in plant breeding is to be expected5. Lastly, the method does not seem to be promising in theory but has in the past few years already given rise to a number of agronomically significant varieties (3,220 cultivars), in over 220 crop species6.

Hall et al.7 further stressed that when incorporating recessive traits, backcross breeding can be slow or require considerable effort. For these traits, mutation breeding can be effective, especially if the trait is easy to screen. The greatest significance of mutations lies in the fact that it can create something de-novo that did not exist before. It also provides alleles that are required for various types of genetic studies of populations8. Fawole9 among many other authors, stressed the possibility of using the mutants in cultivar improvement, physiological studies and the development of a genetic linkage map for cowpea. Therefore, the objective of this study was to select the mutant lines that possess useful traits that could be added to the gene pool of this economically important crop.

MATERIALS AND METHODS

Mutagenesis and generation of mutant population: The eight cowpea accessions used in this study were the cultivars IB, IB-BPC, IB-Y-1 and IB-CR and elite cultivars IT86D-719, IT86D-1010, IT894KD-374-57 and IT90K-284-2. Details on the sources, characteristics of the cowpea accessions used, mutagenesis procedures by gamma ray and generation of M1 popula2tions have been described in Olasupo et al.10. Matured and dried pods were harvested in May, 2013 from M1 plants across the treatments into separate envelopes and stored until they were later advanced to M2 generation in September, being the late planting season of 2013. The seeds produced by M2 plants were harvested in December, 2013 and were later advanced to M3 and M4 generations in the screen house in February, 2014 and March, 2015, respectively.

Screening for gamma induced mutants and phenotyping in the M2 generation: Screening for morphological mutants of cowpea in the M2 generation was carried out on the field at the Teaching and Research Farm of the University of Ibadan. The number of seeds planted to each treatment in the M2 generation (Table 1) was determined by the number of seeds harvested from M1 plants. For all the treatments, field plantings were made at the spacing of 60 cm between rows and 30 cm within the rows. Screening for mutant in each treatment was carried out by scoring the M2 plants from germination to maturity for any change in phenotype observed when compared with the parent plants (control treatment).

Table 1:
Quantity of cowpea seeds planted for gamma induced mutation screening at M2 generation

Suspected mutants at seedling/early growth stage were carefully uprooted with the aid of a hand-trowel, transplanted into plastic pots filled with sterilized soil and transferred to the screen for adequate care. Selections of putative mutants were made progressively from seedling stage to the maturity stage of the plants on field by their phenotypic traits. Seeds obtained from these putative mutants were later advanced to M4 generation in the screen house to confirm their mutant phenotypes when compared with their parents. Descriptive statistics was used to analyze the data. Mutation frequency (rate) per radiation level was calculated as the total number of mutants occurring in 100 plants.

RESULTS

Screening and selection for mutant phenotypes in the M2 and M3 generations were based on the observed phenotypic changes and deviation from the phenotypes of each parent. Diverse mutations spectra were selected in the M2 plant population (Table 2). Mutants were selected by observed variation in their morphological traits in the M2 generation across radiation treatments.

No definite pattern was observed in the mutation frequencies across gamma radiation treatments in all the cowpea accessions. High frequencies of yellow and white seedling (albino) mutants (Fig. 1) were observed in most of the treatments except in all the control treatments and few among the radiation treatments. These albino seedlings were chlorophyll deficient lethal mutants that died a week after germination.

Burnt leaf mutants (Fig. 2) were observed in the mutagenic sub-population of IT86D-719 with 100 and 300 Gy treatments. The mutants were characterized by leaves that appear as if leaves have been partially burnt in fire flame. The burnt leaf mutation affects the upper surface feature and colour of the leaves which is partially folded at the margin and pale green in contrast to the straight-smooth margin and normal green colour of the parent. These mutants became stable at M3 generation.

Fig. 1:
Albino (white and yellow) seedling mutants

Fig. 2:
Burnt leaf mutant of IT86D-719 (IT-719BN-1)

Table 2:
Spectra and frequencies of mutation in the M2 generation following 60Co gamma irradiation of cowpea seeds at different dosage levels
AS: Albino seedling, BL: Burnt leaf, FPL: Four-primary leaf, TPL: Three-primary leaf, YL: Yellow leaf, V: Variegated leaf, SP: Short pod, NPU: Non-petiolate unifoliate, NL: Narrow leaf, ET: Erect-tall, BS: Big seed, LTL: Lettuce leaf, MS: Male sterile, SL: Serrated leaf, SLY: Small leaf yellow, YD: Yellow dwarf, NPTL: Non-petiolate terminal leaflet, DC: Dwarf crinkled, NS: No survived plant

Three seedlings which produced four-primary-leaf mutants were observed in the treatment (300 Gy) of IT86D-719 and IT90K-284-2 (Fig. 3). The three mutants (IT-719FPL-1, IT-719FPL-2Fas and IT-284-FPL) were selected at M2 generation on the field and bred true at M3. The secondary leaves subsequently produced by these mutants were normal trifoliate (Fig. 3).

In addition to the four-primary leaves produced by IT-719FPL-2Fas, the mutant also produced fasciated stem (Fig. 4). The fasciated stems of this mutant were characterized with opposite nodes as opposed to alternate nodes produced by the parent and between one and two leaves were produced at each node of the mutant. The mutant grew with higher vigor and biomass when compared with the parent.

Fig. 3(a-b):
Four-primary leaf mutant produced from IT86D-719, (a) Parent and mutant seedlings at germination and (b) The mutant producing normal trifoliate secondary leaves

Fig. 4(a-c):
Four-primary leaf mutant (IT-719FPL-2Fas) showing fasciated stem trait, (a) Fasciated stem mutant with vigorous stem as compared to the parent, (b) Fasciated stem mutant producing multiple leaves at opposite nodes and (c) Fasciated stem mutant producing fasciated peduncle

A cross between this mutant and IT90K-284-2 produced fasciated stem M2 plants with multiple leaves at opposite nodes and another plant with fasciated peduncle which yielded up to 12 pods on the peduncle (Fig. 4).

The four-primary leaves mutant (Fig. 5), IT-719FPL-2Fas also produced flowers with four standard petals instead of the normal two fused standard petals of cowpea. Dissected flowers of these mutants revealed the presence of extra stamens and carpel (Fig. 6).

Fig. 5(a-b):
Mutant flower of IT90K-FPL-2 (a) Normal cowpea flower showing double standard petal flower and (b) Mutant flower producing four standard petals

Fig. 6(a-b):
Dissected mutant flowers of IT90K-FPL-2, (a) Flowers producing one and two staminates and (b) Flowers producing one, two and three carpel

Table 3:
Types and frequencies of the floral parts produced by IT90K-FPL-2 cowpea mutant

Observation on sampled population of this mutant showed the plants produced five types of flowers that are characterized with varied number of floral parts (Table 3). Each of the mutant flowers produced 12 stamens which are distributed into fused stamen (9-10 in number) and staminate (1-3 in number), whereas, the parent flowers produced 10 stamens. The variation observed in the number of stamens was such that an increase in the number of staminate compensated for a reduction of the number of fused stamen. In addition to these, the mutant flowers were observed with one, two and three carpel.

Some seedling mutants with three primary leaves at germination (Fig. 7) were observed at M2 generation in gamma irradiated IB-BPC, IT86D-719, IT86D-1010, IT89KD-374-57 and IT90K-284-2 (Table 2).

Fig. 7:
Three primary leaf seedling mutant

Fig. 8:
Yellow leaf mutant (IT86D-719Y)

However, none of the three primary leaf mutations was stable but all these mutations were reverted back to normal seedlings with two primary leaves at M3 generation.

Yellow leaf mutants (yellow-flush) were observed and selected from gamma irradiated cowpea IT86D-719 and IT89KD-374-57 at M2 generation (Table 2). However, only one of the yellow mutants selected from IT86D-719 treated with 200 Gy radiation (IT86D-719Y) became stable at M3 generation. This mutant produced leaves with yellow colour which appears prominently at flushing and faded as the leaves grow older (Fig. 8).

Variegated leaf mutant (Fig. 9) was only observed and selected in IB-CR at radiation level of 300 Gy. The leaf variegation in this mutant was not spread over but limited to a branch of the plant. A variegated pod produced from this branch generated seeds which were planted out at M3. However, some of the seedlings generated from the variegated pod were devoid of chlorophyll and did not survive while others seeds harvested from the mutant produced M3 plants that lack variegated leaf trait.

The non-petiolate unifoliolate mutant (Fig. 10) was selected in IT89KD-374-57 at the treatment level of 400 Gy. This mutant produced non-petiolate unifoliate (single) leaves instead of the normal trifoliate leaves of cowpea. The mutant also produced flower buds that were arranged on peduncle like rose bud. However, the flower buds produced by this mutant were unable to open and consequently no seed was produce by this mutant.

A narrow leaf seedling mutant selected in 400 Gy treatment of IT89KD-374-57 is presented in Fig. 11.

Fig. 9(a-b):
Variegated leaf mutant from IB-CR, (a) Variegated leaves of the mutant and (b) Variegated pod produced from variegated branch of the mutant

Fig. 10(a-b):
Mutant from IT89KD-347-57, (a) Non-petiolate unifoliate mutant and (b) Non-petiolate unifoliate mutant producing abnormal flower buds

Fig. 11:
Narrow leaf seedling mutant (IT89KD-NL)

Apart from the narrow leaf trait of the seedling’s primary leaves, the plant produced petiolate leaves with unstable leaflet number which ranged between unifoliate and trifoliate (Fig. 12). The mutant plant became stable with respect to these traits at M3 generation.

Fig. 12:
Petiolate unifoliate, difoliate and trifoliate produced by narrow leaf seedling mutant (IT89KD-NL)

Fig. 13:
Erect tall mutant (IB-ER) from Ife brown cowpea cultivar

Fig. 14:
Lettuce leaf mutant (IB-LT)

Another erect tall mutant cowpea was selected from the M2 sub-population of IB at the treatment level of 100 Gy. This mutant was erect, tall, non-branching with raised peduncles (Fig. 13) as opposed to the parent which is semi-erect with many spreading branches. The mutant was stable for these traits at M3 generation.

Fig. 15:
Small leaf yellow mutant IT-719G200SLY

The lettuce leaf mutant selected from M2 population of IB-CR at radiation level of 100 Gy is presented in Fig. 14. This mutant (IB-LT) has pale green twisted leaves traits that make it appear like lettuce plant in contrast to the crinkled leaf of the parent.

Two small leaf yellow mutants cowpea were selected at M2 generation from IT86D-719 at treatment level of 200 Gy. One of these plants (IT-719G200SLY) produced small yellow trifoliate leaves (Fig. 15), flowers with short style (Fig. 16) and set seeds at maturity, while the other mutant plant produced small deformed flowers and did not set seed.

Three big seed mutants were also selected from IT90K-284-2 at the treatment levels of 100, 200 and 300 Gy (Table 2). These mutants were characterized with bigger seeds when compared to the seeds produced by the parent (Fig. 17). They were selected at M2 and became stable for this trait at M3 generation.

Observed frequencies of all cowpea mutants selected in this study are presented in Table 2. Cowpea mutants with novel phenotypes were selected from five out of the eight accessions used in this study. No definite trend was observed in the mutation frequencies with respect to radiation treatments in this study. However, higher range of mutation frequencies (0.0057<1.745) was observed among the four elite cultivars (IT86D-719, IT86D-1010, IT86KD-374-57 and IT90K-284-2) when compared with low frequencies range (0<0.4013) observed among IB and its derivatives at the radiation dosages sub-ministered. Albino mutants (yellow and white seedlings) were observed in higher numbers than other mutants in all the cowpea accessions and across radiation treatments.

DISCUSSION

One of the benefits of induce mutation is that it is used to create genetic variations and provides the raw materials for genetic studies and for the breeders to develop new varieties of plants and animals. Some cowpea mutants with novel phenotypes selected from five out of the eight accessions used in this study can be phenotypically classified into macro mutations and micro mutations.

Fig. 16:
Small leaf yellow mutant flower with short style

The burnt leaf, four primary leaf, yellow leaf, lettuce leaf, narrow leaf, double standard petal and erect non-branching mutant traits selected in the present study may be classified as macro mutations. Easily detectable mutant traits are phenotypically visible and morphologically distinct with qualitative inherited genetic changes11. Such observable changes occur as a result of the effect of few major genes or oligogenes yielding macro mutations. Horn et al.12 reported some macro mutations affecting flower and seed color. Micro mutations on the other hand, are the result of polygenes each with minor genetic effect showing quantitative inheritance which can be detected only by the help of statistical methods and quantitatively inherited genetic parameters12,13. Thus micro mutations identified among selected mutants in this study are those with big seed and short pod traits.

The yellow and white seedling (albino) mutants observed at high frequencies in most treatments were chlorophyll deficient. Lack of chlorophyll in the primary leaves and stem produced lethal effect on these albino seedlings shortly after germination. High frequencies of mutations such as those observed for the albino trait have been attributed to the action of transposable elements14 and gamma rays have been reported to stimulate transposon activity15. Loss of these albino mutants at the seedling stage did not allow further study to be conducted on them. However, several authors have reported on the inheritance of mutations resulting in chlorophyll deficiency in cowpea. Kirchhoff et al.16 concluded that chlorophyll deficiency in cowpea mutants was controlled by single recessive gene.

Fig. 17(a-d):
Big seed mutants, (a) Parent seed, (b) Big seed mutant IT90K-BS-1, (c) Big seed mutant IT90K-BS-3 and (d) Big seed mutant IT90K-BS-4

The tall-erect non-branching cowpea mutant (IB-ER) could be a useful mutant to breed for tall and erect cowpea varieties that may be used for mechanized farming. The big seed mutants (IT90K-BS-1, IT90K-BS-3 and IT90K-BS-4) selected possess morpho-agronomic characters that could be used for the improvement of cowpea production. Fasciated stem mutants (IT-719FLP-2Fas), because of their vigor and numerous leaf productions, have the potential to be used as fodder crop or introgressed for dual purpose cowpea breeding programme. Excess of floral parts produced by this mutant was as result of the changes in its genome caused by gamma irradiation. Two and three carpel found in its flower implies that a cowpea variety with twin and triplet pods could be developed using this mutant line. Porbeni17 earlier reported a twin pod that arose from fasciated stem cowpea mutant having two or more styles. Variation observed in the number of floral parts of this mutant appears to be due to the action of transposable element in cowpea. Flower mutations induced by transposable element in cowpea in which both mutant and wild type flowers were found on the same plants had been reported by Oluwatosin18. In addition to these, numerous pods produced on fasciated peduncle of the fasciated stem mutant (IT-719FLP-2Fas) and the big seed mutants (IT90K-BS-1, IT90K-BS-3 and IT90K-BS-4) selected possess morpho-agronomic characters that could be used for cowpea improvement. The four- primary leaf mutants (IT-719FLP-1 and IT-719FLP-2Fas) and narrow leaf seedling mutant (IT89KD-NL) could be used as genetic markers at seedling stage of plant growth. Burnt leaf mutants (IT-719BN-1 and IT-719BN-2), double standard petal flower mutant (IT-719FLP-2Fas) and lettuce leaf mutant (IB-LT) could also be useful as genetic markers. Yellow leaf mutants (IT-719Y and IT-719SLY) could be used to develop plant with aesthetic value. Porbeni17 selected some cowpea lines for their ornamental potentials. Diverse and genetically stable novel mutants of cowpea selected in this study shows the utility of induce mutation for creating new genetic variability for the purpose of broadening genetic diversity and increasing germplasm collection of crop plants.

The frequencies of yellow and white seedling (albino) mutants were higher than other mutants in all cowpea accessions and across radiation treatments in this study. These albino seedlings were lethal mutants because these seedlings were devoid of chlorophyll needed for photosynthesis. Among many authors, Olasupo19 and Sangsiri et al.20 reported similar observations from cowpea seeds treated with ethyl methane sulfurnate (EMS) and mungbean seed treated gamma ray, respectively. The three-primary leaf mutants selected could not breed true at M3 generation probably due to reversion of the mutation. It indicates that there is genetic instability in the gene controlling three-primary leaf trait in cowpea.

The higher mutation rate observed in the elite cultivars (IT86D-719, IT86D-1010, IT86KD-374-57 and IT90K-284-2) than Ife brown (IB) and its derivatives suggest that the elite cowpea lines possess more mutable sites in their genomes than IB and its derivatives. Several authors had suggested that this difference is related to the genetic background or constitution of test varieties21,22. Researchers therefore, proposed among other factors, that mutability of a gene may also be influenced by inherent genetic system remaining in an organism. The IT86D-719, IT86D-1010, IT86KD-374-57 and IT90K-284-2 were advanced cultivars which makes their genes more prone to mutation than IB and its derivatives. However, no definite trend was observed in the rates of mutant phenotypes across radiation treatments in all the cowpea accessions studied. This indicates that mutations are random events and that mutation induction rate in cowpea appears to be determined by genetic background of induced plant genotype.

CONCLUSION

Induced mutation is a valuable tool for creating new genetic variability to complement existing germplasm. The present study focused at selecting new mutants of cowpea with novel morphological and agronomic traits. The elite cowpea cultivars studied appeared to have more mutable sites than IB and its derivatives. Several mutants selected are characterized by useful and interesting traits confirming induce mutation technique as a valuable tool for crop development.

SIGNIFICANCE STATEMENT

This study revealed the evolution of new variants of cowpea species and consequence increase in its genetic base by induced mutations. New mutant lines selected possess diverse morpho-agronomic traits that were not in existence before, making more alleles available and these are to be utilized by plant breeders for cowpea improvement. The new mutants developed in this study will also be useful tools to plant geneticist for further genetic analysis, linkage mapping and physiological studies.

ACKNOWLEDGMENTS

Authors would like to thank the management of University of Ibadan, Nigeria for the provision of facilities at the Teaching and Research Farm, University of Ibadan to carry out this study. We also acknowledge the good support of the field staff of Teaching and Research Farm during this study.

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