Keywords

15.1 Introduction

Cowpea [Vigna unguiculata (L.) Walp.] is a legume crop cultivated worldwide as pulse, vegetable, forage, green manure and cover crop (Smartt 1990). Due to its high protein content in leaves, pods and grains, it is widely regarded as “poor man’s meat” (Boukar et al. 2018). The primary centre of origin is Africa because it has high genetic diversity there. Cowpea can be grown easily in low fertility soils (Eloward and Hall 1987) and has the ability to fix atmospheric nitrogen like many other legumes (Ehlers and Hall 1996). Cowpea is one of the most tolerant legumes to drought because of its ability to grow in areas without irrigation and irregular rainfall (Agbicodo et al. 2009). It is one of the best crops that fit well in rice-wheat cropping systems.

The cowpea seed contains protein (23–32%), carbohydrate (17.50–60%) (Khalid and Elharadallou 2013; Kirse and Karklina 2015) and fat (1%) (Kirse and Karklina 2015) on dry weight basis. Compared to cereal and tuber crops, two- to fourfold more protein is present in cowpea (Sebetha et al. 2014; Trehan et al. 2015). Apart from this, it also contains soluble and insoluble fibre, phenolic compounds, minerals and B group vitamins along with many other functional compounds which are health promoting (Mudryj et al. 2012; Liyanage et al. 2014) The tender green pods of cowpea are rich in crude protein (3.2%), iron (2.5 mg per 100 g), calcium (80 mg per 100 g), phosphorus (74 mg per 100 g), vitamin A (941 IU per 100 g), vitamin C (13 mg per 100 g) and dietary fibre (2 g per 100 g), making it an excellent vegetable (Singh et al. 2001).

The worldwide production of pulse cowpea is 7.41 million tonnes cultivated in an area of 12.58 million hectares with an average productivity of 589 kg/ha. The leading cowpea-producing countries (Table 15.1) are Nigeria (340,992 tonnes) followed by Niger (1,959,082 tonnes) grown in an area of 3,782,760 ha and 5,178,517 ha, respectively. In terms of productivity (Table 15.2), leading countries are Palestine (3929.40 kg/ha) followed by Egypt (3677.20 kg/ha) (FAOSTAT 2019).

Table 15.1 Leading cowpea-producing countries in the world (FAOSTAT 2019)
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Table 15.2 Top five countries leading in cowpea productivity (FAOSTAT 2019)
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Accelerated development of varieties should be combined with speedy dissemination of developed varieties and agile withdrawal of obsolete varieties. To reduce the risk of obsolete varieties which were developed a decade ago in a different climate than today’s scenario should be replaced with varieties developed within one decade. To achieve this cowpea breeding system is to be strengthened with free international exchange of germplasm, elite varieties, speed breeding, increasing the selection intensity, large-scale phenotyping and marker- and genomics-assisted selection for accuracy (Atlin et al. 2017).

15.2 Genetic Diversity and Taxonomy

Large amounts of cowpea landraces and cultivated cowpeas were present in West and Central Africa (Padulosi and Ng 1997) which is considered as the centre of origin of cowpea. Different organizations of the world hold around 36,383 cowpea germplasm (Table 15.3) under ex situ conservation (Dumet and Fatokun 2010). Apart from this, the National Bureau of Plant Genetic Resources, New Delhi, holds 5000 germplasm of cowpea under ex situ condition as exhibited in cowpea germplasm field day held on 22 October 2019. Out of the total germplasm stored under ex situ condition, the majority (60%) of the accessions were farmers’ varieties/landraces, 5.2% are breeding lines, 2.0% are wild and the remaining up to 31% that were unknown are not documented (Dumet and Fatokun 2010).

Table 15.3 Number of cowpea accessions reported from various international holders under ex situ condition
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Based on the characteristics of pod, seed and ovule, the cultivated types of cowpea (Table 15.4) have been divided into five cultivar groups (Pasquet 1998, 1999). Among them unguiculata is the largest cultivar group. The vegetable cowpea cultivar group sesquipedalis (also known as yardlong bean, asparagus bean, snake bean and long bean) has more than 16 ovules and seeds spaced apart within the pod (OECD 2016).

Table 15.4 The five cultivar groups of cultivated cowpea V. unguiculata ssp. unguiculata (OECD 2016)
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The Vigna unguiculata subspecies cultivated in India were V. unguiculata ssp. unguiculata and V. unguiculata ssp. biflora grown predominantly for pulse purpose, whereas V. unguiculata ssp. sesquipedalis (yardlong bean) is grown for its immature pods as vegetable. The vegetable cowpea is grown widely in India, China, Sri Lanka, Bangladesh, Indonesia and the Philippines (Pant et al. 1982; Chakraborti 1986; OECD 2016).

The classification and nomenclature of Vigna unguiculata species complex was done by several workers, viz. Verdcourt (1970), Marechal et al. (1978), Mithen and Kebblewhite (1993), Padulosi (1993) and Pasquet (1993/1998). Presently the Vigna unguiculata species complex has been divided into 11 subspecies (Padulosi 1993; Pasquet 1993a, b, 1997; Padulosi and Ng 1997) (Table 15.5). There exists a varying degree of crossability of the ten wild subspecies with the sole cultivated cowpea subspecies. The subspecies dekindtiana, alba, tenuis (and var. spontanea), stenophylla and pubescens were previously under dekindtiana subspecies, so-called conveniently as dekindtiana group. The subspecies baoulensis, letouzeyi, burundiensis, pawekiae and aduensis were previously under subspecies mensensis and conveniently called as mensensis group. The cultivated cowpea along with dekindtiana group was highly self-pollinated, whereas the mensensis group was cross-pollinated (OECD 2016).

Table 15.5 Classification of Vigna unguiculata (L.) Walp. and its subspecies complex (Pasquet 1993a, b, 1997)
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The two botanical varieties of annual cowpea are Vigna unguiculata unguiculata var. unguiculata which is cultivated and V.u.u var. spontanea which is a wild form. The immediate progenitor of the cultivated cowpea is V. unguiculata ssp. dekindtiana sensu Verdc (V. unguiculata var. spontanea (Schweinf.) Pasquet) (Padulosi and Ng 1997).

15.3 Genetics

Cowpea is a diploid with a chromosome number of 2n = 22. Genetics of cowpea were reviewed comprehensively by Fery (1980, 1985), Fery and Singh (1997), Singh (2002) and Boukar et al. (2018). The genetic control of various traits was presented (Table 15.6).

Table 15.6 Genetic control of various traits in cowpea
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In vegetable cowpea breeding, both additive and dominance variances control the trait expression. High amount of variance was observed for number of pods per plant, pod yield, pod length and crude fibre content (Subbiah et al. 2013). Genetic analysis studies had shown that in vegetable cowpea, number of clusters per plant had high additive and additive × additive genetic component, while the pod weight had high broad and narrow-sense heritability suggesting that these traits should be focused during early generation selection. Selection for pod yield should be done in later generations, and for multilocation testing of yield stability number of pods per plant may be used as a criterion (Pathmanathan et al. 1997). Green pod yield per plant showed positive significant correlation with pod length, ten pod weight and number of seeds per pod. The path coefficient analysis indicated that the highest positive direct effect on green pod yield per plant was exhibited by the number of green pods per plant followed by days to 50% flowering, ash content and pod length (Hitiksha et al. 2014).

An effective cowpea breeding strategy involves combining the erect, determinate and early maturing characters of cv. Unguiculata (ssp. unguiculata) or Biflora (ssp. cylindrica) genotypes with the long, succulent and fleshy podded characters of cv. Sesquipedalis (ssp. sesquipedalis) genotypes. Crossing between genotypes of sesquipedalis and those of unguiculata and cylindrica revealed low success due to specific cross combinations, genetic divergence and environment. Additive genetic variance was predominant for pod length and weight and protein content in pods and seeds. Selection in the advanced generations should be based on bushy or less viny, high-yielding segregates with appreciable protein contents in pods and seeds (Hazra et al. 2007).

15.4 Improved Varieties of Cowpea

The International Institute for Tropical Agriculture (IITA) developed several pulse-type cowpea varieties (Table 15.7) with high yield ranging from 1.5 to 2 tonnes per hectare. The improved varieties of IITA viz., IT-16 (1400 kg/ha), IT-18 (1510 kg/ha), IT-04 K-321-2 (1460 kg/ha), IT-97 K-390-2 (1370 kg/ha) and IT-99 K-494-4 (1660 kg/ha) matures in about 90-94 days and are tolerant to drought, leaf spot and bacterial diseases and have a reddish-brown seed colour. All these IITA developed varieties have protein content of more than 25% (Lopez 2019). The variety IT99K-494-6 is an Alectra-resistant variety (Boukar et al. 2012). The pulse type of cowpea gives a maximum yield of 1.5 to 2.0 tonnes per hectare, whereas by cultivating vegetable cowpea bush varieties (Table 15.8), the maximum yield of up to 15–18 tonnes per hectare can be taken in 6–8 pickings based on the variety cultivated. But for cultivating vegetable cowpea, irrigation is required at regular intervals, and the first harvest of stringless tender pods is taken 55 days after sowing.

Table 15.7 Improved varieties of cowpea by IITA
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Table 15.8 Improved varieties of vegetable cowpea in India
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15.5 Breeding Cowpea for Pest Resistance

15.5.1 Cowpea Golden Mosaic Disease Resistance

In cowpea, infections caused by viruses are the most important as they can reduce the production from 60% to 80% in susceptible varieties. Among them, cowpea golden mosaic disease (CPGMD) is of prime importance causing extensive losses of 40–78% in production (Santos and Freire-Filho 1984). This disease is caused by begomovirus of the Geminiviridae family. The main symptom (Fig. 15.1) was golden mosaic of the leaves which then coalesces and cause complete yellowing of the leaves. The vector for transmission is whitefly. Resistance to CPGMD is attributed to two dominant and independent genes (Sangwan and Rish 2004) and single dominant gene (Kumar et al. 1994; Rodrigues et al. 2012). In Brazil, three AFLP markers, E.AAC/M.CCC515, E.AGG/M.CTT280 and E.AAA/M.CAG352, were found linked to CGMV resistance gene at 50.4, 24.4 and 28.7 LOD scores, respectively (Rodrigues et al. 2012). The cowpea golden mosaic DNA A virus isolates from India and Nigeria has similarity of only 62% which indicates that there exists a great viral diversity in cowpea golden mosaic virus isolates globally (Winter et al. 2002).

Fig. 15.1
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Symptoms of cowpea golden mosaic disease on cowpea line VRCP-195-2

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To identify the cowpea genes that confer durable resistance to CPGMD , we should use defined gemini virus isolates for controlled inoculation of indicator cowpea genotypes where it produces typical golden mosaic symptoms consistently in proven susceptible genotypes and no symptoms in resistance genotypes (Singh et al. 1997). Another feasible method for transmission of the virus is by grafting the diseased plant scion onto host plant root stock by top cleft or side cleft grafting. For better success, the rootstock and scion should be of similar thickness (Green 1991).

15.5.2 Cercospora Resistance

In humid tropics, Cercospora leaf spot (CLS) (Fig. 15.2) is an important disease of cowpea causing a yield loss from 36% to 42% (Schneider et al. 1976; Fery et al. 1977). Cercospora leaf spot-causing pathogens in cowpea are Pseudocercospora cruenta (Deighton 1976) and Cercospora apii s. lat. emend. (Crous and Braun 2003). Booker and Umaharan (2008) developed four crosses from the above four resistant genotypes and two susceptible genotypes CB27 and Los Banos Bush Sitao no.1 and developed six populations (Parent 1, Parent 2, F1, F2, BC1 and BC2) for each cross combination to know the genetics of inheritance to Cercospora leaf spot disease caused by Pseudocercospora cruenta in cowpea. He also observed that there was a differential resistance to both the pathogens among the tested cowpea varieties. For P. cruenta alone, four genotypes, VRB-10, IT-86D-719, IT87D-939-1 and IT-87D-792, were found resistant. Booker and Umaharan (2008) developed four crosses from the above four resistant genotypes and two susceptible genotypes CB27 and Los Banos Bush Sitao no.1 and developed six populations (Parent 1, Parent 2, F1, F2, BC1 and BC2) from each cross to know the genetics of inheritance to Cercospora leaf spot disease caused by Pseudocercospora cruenta in cowpea. These populations were screened under induced epiphytotic conditions in four separate field experiments. The onset of CLS disease varied from 35 to 48 days after sowing. The results from this study showed that resistance to CLS is governed by genetic mechanisms varying from monogenic, oligogenic to polygenic inheritance. In the cross CB27 × IT86D-719, intermediate level of resistance was found in F1 generation, and normal distribution was observed in F2 generation for CLS disease which confers polygenic resistance. Oligogenic inheritance was observed in other three crosses. In the cross CB27 × IT87D-939-1, single gene model with incomplete dominance was observed followed by single gene model with complete dominance in the cross CB27 × VRB-10. A trigger model was observed in the cross Los Banos Bush Sitao × IT86D-792 where three major genes were involved. In all these crosses, the role of minor genes was also observed. Based on symptomatic to non-symptomatic plants’ ratio, these probable inheritance mechanisms were observed.

Fig. 15.2
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Cercospora infestation on leaves of CP2 vegetable cowpea variety

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15.5.3 Anthracnose Resistance

In cowpea, anthracnose is caused by Colletotrichum lindemuthianum which is one of the destructive diseases. Field cowpeas (Vigna unguiculata ssp. cylindrica) show various levels of resistance to this disease, whereas vegetable-type cowpeas (Vigna unguiculata ssp. sesquipedalis) are highly susceptible to this disease. The linked markers identified for this disease are ISSR primers UBC 810 and UBC 811 which have yielded markers at 1.4 and 1.5 kb in resistant genomes, respectively, whereas RAPD primer OPA02 has yielded a marker at 850 bp in susceptible genome (Pradhan et al. 2018). In cowpea, the genetics of anthracnose resistance is not reported, while in various legumes, the gene action was reported and confusing. Polygenic resistance to anthracnose was reported in common bean (Sousa et al. 2014), and the genes offering resistance were fine mapped (Sousa et al. 2015). In lupin, single dominant gene has conferred resistance to anthracnose (Yang et al. 2012).

15.5.4 Bruchid Resistance

The main storage pest of cowpea causing considerable loss is cowpea seed beetle (Callosobruchus maculatus (P.)) commonly known as bruchid. Apart from seed loss, it reduces the seed quality and affects germination. The bruchid resistance is characterized by delayed and staggered infestation along with lower bruchid emergence (Singh and Singh 1989). It was observed that after infestation of 200 g cowpea seed sample in different cowpea varieties with 2 pairs of bruchid had 25–26% seeds damaged in resistant lines, while there was 95% damaged seeds in susceptible variety after storing for 103 days (Singh et al. 1985). The bruchid resistance in cowpea is governed by two pairs of recessive genes which showed that any outcrossing reduces the resistant plants’ proportion in the succeeding generation. The line Tvu 2027 was identified as moderately resistant to bruchids. Apart from this, IT84S-2246-4 is another important line which has combined resistance to bruchids, aphids and thrips along with resistance to ten diseases. For bruchid resistant breeding plants should be selected in F2 based on plant type, maturity, seed type and resistance to diseases, and then the F3 seed from individual plant progeny of each F2 plant was tested for bruchid and aphid resistance. Then the selected progenies from subsequent F4, F5 and F6 generations were selected for insect and disease resistance along with yield (Singh and Singh 1985). A number of Vigna species were also screened for resistance to Callosobruchus maculates and were found that V. luteola and V. adenantha were immune and V. oblongifolia and V. racemosa were moderately resistant (Ofuya 1987). The most of these Vigna species do not cross with cultivated Vigna.

15.5.5 Pod Borer Resistance

Maruca vitrata also called as legume pod borer is an important cowpea pest that causes huge yield losses between 20% and 80% if no control measures are employed. The larva of Maruca is the most destructive stage that causes damage mainly during reproductive stage of the plant by feeding on the young shoots, floral parts, pods and seeds. In comparison with any other insect pests of cowpea, Maruca causes higher yield loss (Fatokun 2009). Through conventional breeding, varieties resistant to aphids and thrips and low levels of resistance to storage weevil were developed, less progress was observed while breeding resistance to Maruca in cowpea. After screening several cowpea accessions along with their wild relatives, it was found that Vigna vexillata accessions have resistance to Maruca vitrata (Fatokun 2009). Strong cross-incompatibility exists between V. vexillata and V. unguiculata, making the gene transfer impossible (Fatokun 2009). The best alternative is development of transgenic cowpea against legume pod borer by using crystal proteins (Cry) and vegetative insecticidal proteins (Vips) of the Bacillus thuringiensis (Bt) bacterium (Bett et al. 2017). Five Vip genes, vip3Aa35, vip3Af1, vip3Ag, vip3Ca2 and vip3Ba1, for resistance to Maruca pod borer were identified, cloned and over-expressed in Escherichia coli to produce Vip3 protein. Among these Vip3Ba1 proteins was selected as a candidate gene for cowpea transformation because of its effective larval growth inhibition. Transgenic lines with Vip3Ba protein expression were found completely free from Maruca pod borer in insect feeding trials. From this, it was proposed that combining existing cry-transgenic cowpea and vip-transgenic cowpea will provide additional resistance and the greatly delay the resistance development by Maruca (Bett et al. 2017).

To know the genetics of transgenic cowpea carrying Cry1Ab transgene, two lines of transgenic cowpea (TCL-709 and TCL-711) containing transgene Cry1Ab were crossed with three traditional cowpea genotypes (IT97K-499-35, IT93K-693-2 and IT86D-1010) and found monogenic segregation in F2 and BC1 with 3:1 and 1:1, respectively, by using Bt strips analysis and also by artificial infestation of legume pod borer. As there was stable transmission in sexual generations of cry-transgenic cowpea under lab and field conditions, transgenic cowpea varieties for insect resistance can be developed by combining conventional breeding with marker-assisted selection (Mohammed et al. 2015).

First genetically modified cowpea resistant to pod borer was introduced in Nigeria in 2011 (Klopez 2009; Abutu 2017) and then to Burkina Faso, Ghana and Malawi (Gomes et al. 2019). The Nigerian Biosafety Management Authority (NBMA) approved the commercial release of GM cowpea on 29 January 2019 to Nigeria farmers which facilitated the release of Pod Borer-Resistant Cowpea (PBR Cowpea)-event AAT709A (Lopez 2019).

15.6 Tissue Culture Plant Regeneration Protocols for Cowpea

In many tropical legumes, limited transformation protocols were reported due to their regeneration inability under tissue culture conditions (Somers et al. 2003). As phenolic levels are high that lead to explants’ oxidation, the Leguminosae family is highly recalcitrant (Anthony et al. 1999). In spite of several numerous protocols for cowpea in vitro regeneration, there was no efficient protocol in vitro regeneration due to difficulty in reproducibility and very low regeneration frequency (Anand et al. 2000).

Raveendar et al. (2009) developed a rapid highly efficient system of organogenesis in cowpea, where the seeds were pretreated for 3 days with 13.3 μM BAP and were cultured for 2–3 weeks on MSB5 medium supplemented with 6.6 μM BAP for induction of multiple shoot buds. The multiple shoot buds were transferred onto a 0.5 μM BAP amended medium for shoot elongation. On a growth regulator-free medium, the elongated shoots were rooted and then the plantlets were transferred to soil after 12 days, with a survival success of 90-95%. Here MS medium (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins (MSB5) containing 3% (w/v) sucrose and 0.7% agar supplemented with growth regulators was used. The pH of the medium was adjusted to 5.8 by using 1 M NaOH or 1 M HCl and autoclaved at 1.06 kg cm−2 at 121 °C for 15 minutes. The incubation conditions for the culture include 25 ± 2 °C with irradiance of 50 μmol m−2 s−1 with 16 hours of photoperiod and 55% relative humidity.

15.7 Embryo Rescue

For Vigna species, the medium containing MS basal nutrients (Murashige and Skoog 1962) with sucrose (88 mM), casein hydrolysate (500 mg L−1) and agar (8 g L−1), but devoid of plant growth regulators (EGM), was found to be the best medium for successful germination of immature embryos in four Vigna species, Vigna vexillata, V. lanceolata, V. marina, V. luteola, and two mung bean subspecies, V. radiata ssp. radiata and V. radiata ssp. sublobata (Palmer et al. 2002).

15.8 Genomics-Assisted Breeding

The integration of new technologies into public plant breeding programs can make a powerful step change in agricultural productivity when aligned with principles of quantitative and Mendelian genetics (Cobb et al. 2019). Cowpea (Vigna unguiculata L.) has a chromosome number of 2n = 22 and an estimated genome size of 640.6 Mbp (Lonardi et al. 2019). Initially Munoz-Amatriain et al. (2017) developed a highly fragmented draft assemblies and BAC sequence assemblies of cowpea genotype IT97K-499-35, but they lacked completeness required for genome annotation, candidate gene investigation and complete genome comparisons. So, Lonardi et al. (2019) developed an assembly of the single haplotype inbred genome of cowpea genotype IT97K-499-35 by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping and an assembly reconciliation algorithm. Repetitive elements were present in about half of the sequences assembled in cowpea that propound that differences among genome size of Vigna species were mainly due to the changes in Gypsy retrotransposon quantity. Based on synteny with common bean (Phaseolus vulgaris), revised chromosome numbering has been adopted for cowpea chromosomes (Lonardi et al. 2019).

Molecular markers permit the indirect selection for desired alleles of genes of interest, independent of the conditions and stage of crop growth (Moose and Mumm 2008). Markers were adopted for breeding in cowpea which includes 1536-SNP GoldenGate assay (Muchero et al. 2009), which has enabled the linkage mapping and QTL analysis by Luca et al. (2011), Muchero et al. (2013) and Pottorff et al. (2014) (Amatriain et al. 2017). Timko et al. (2008) published gene space sequences in IT97K-499-35 genome approximately accounting for 160 Mb. Apart from this, in the software HarvEST:Cowpea, 29,728 unigene sequences were available (harvest.ucr.edu) (Muchero et al. 2009).

15.9 Conclusion

In cowpea, the pedigree selection should be combined with marker-assisted breeding, embryo rescue technology, genomics-assisted breeding and transgenic technology to develop multiple pest-resistant cowpeas in the present-day climate change scenario. Apart from the above technologies used for cowpea improvement, speed breeding technology for cowpea is to be standardized so that 6–8 generations can be taken in a year making the accelerated development of cowpea varieties with improved yield and pest resistance.