Cowpea represents a major food crop for the poor in Asia and especially in Africa.

However, its production is constrained principally by insect pests as well as diseases. Attempts to

improve cowpea insect resistance have not yielded significant result till date. This paper reviews

biotechnological approaches that have been employed to transfer foreign genes into cowpea with a

view to conferring desirable traits on it. The recent advances made in generating cowpea transformants

with stable inheritance of trangenes by the progenies heralds exciting times for the genetic

transformation research on this erstwhile recalcitrant food crop. As such, this genetic transformation

approach could be used to transfer insect resistance traits to the crop species

Obembe, Olawole O.

English

Covenant University Repository

Australian Journal of Basic and Applied Sciences, 3(2): 1083-1086, 2009ISSN 1991-8178© 2009, INSInet PublicationExciting Times for Cowpea Genetic Transformation ResearchOlawole O. ObembeDepartment of Biological Sciences College of Science and Technology Covenant University PMB 1023 Ota, Ogun State, Nigeria.Abstract: Cowpea represents a major food crop for the poor in Asia and especially in Africa.However, its production is constrained principally by insect pests as well as diseases. Attempts toimprove cowpea insect resistance have not yielded significant result till date. This paper reviewsbiotechnological approaches that have been employed to transfer foreign genes into cowpea with aview to conferring desirable traits on it. The recent advances made in generating cowpea transformantswith stable inheritance of trangenes by the progenies heralds exciting times for the genetictransformation research on this erstwhile recalcitrant food crop. As such, this genetic transformationapproach could be used to transfer insect resistance traits to the crop species.Key words: cowpea, genetic transformation, developing countries, food securityINTRODUCTIONThese are exciting times to study genetic transformation of cowpea, using genes that confer desirableagronomic traits to it. For two decades, scientists in this field made great effort with little success to developreliable transformation systems for the crop that is very important as a good source of protein and energy forpeople in developing countries of Africa and Asia. In Africa, it is estimated that over 200 million peopleconsume cowpea daily. Worldwide, cowpea is cultivated on a total area of over 10.5 million hectares, witha total production of 3.9 million tons (FAOSTAT, 2006). In spite of the great importance of this crop to thepeople where it is grown, its productivity is very low due to many biotic and abiotic stress factors. Thesefactors include attack of insect pests and disease pathogens, as well as drought and heat. Although,considerable progress has been made, through conventional genetics and breeding, on cowpea improvementover the decades with respect to resistance to most of these stress factors (Singh, 2007), however, the problemof insect pests still remains largely unresolved. Hence genetic engineering approaches stand out as the onlyeffective alternative means of transferring genes that confer desirable agronomic traits like insect resistance tocowpea. The success of these approaches, however, depends largely on the availability of reliable and efficientmethods of transformation. This review concentrates on advances in cowpea genetic transformation over thepast two decades. It also highlights some implications for food security in the developing countries of sub-Saharan Africa.Early Work on Cowpea Transformation:Garcia’s group in the Netherlands pioneered study on genetic transformation in cowpea. They used anAgrobacterium-mediated system to transform cowpea leaf disc explants (Garcia et al 1986a, 1986b). Althoughstable transformation was achieved in both investigations, as confirmed by Southern analysis, however,transgenic calli failed to regenerate into mature plants.  Similarly, Perkins et al. (1987) and Filippone (1990)were not able to regenerate their transgenic calli that were generated from different explants co-cultivated withAgrobaterium tumefaciens. Grain legumes generally have been regarded as recalcitrant to transformationbecause of poor regeneration ability (especially via callus), and also because in vitro regeneration is genotypespecific. Worst still, most cowpea cultivals are infrequently amenable to regeneration. There is also the problemof compatible gene delivery system. Excellent reviews on legume transformation are also available (Somerset al., 2003, Chandra and Pental, 2003, Popelka et al., 2004).Owing to the failure of callus to regenerate plants, focus was shifted to organogenic regeneration forcowpea transformation. Using longitudinal embryo slices co-cultivated with A. tumefaciens, Penza et al. (1991)Corresponding Author: Olawole O. Obembe, Department of Biological Sciences College of Science andTechnology Covenant University PMB 1023 Ota, Ogun State, Nigeria. Tel:+2348060164341E-mail: odun_wole@yahoo.co.uk.1083Aust. J. Basic & Appl. Sci., 3(2): 1083-1086, 2009reported only transient expression but no evidence of stable integration of the introduced genes. Penza’s groupand another group in the US later separately resorted into exploring the transformability of intact cowpeaembryonic tissues using the electroporation method, using naked DNA in the presence of protectants such asspermine but it was merely transient gene expression that was achieved for all the efforts (Penza et al., 1992,Akella and Lurquin, 1993). Muthukumar’s group also avoided the callus regeneration route by co-culturing de-embryonated cotyledons with A. tumefaciens and selected four plants on hygromycin. Stable transformation wasconfirmed by Southern analysis in only one of the transgenic plants, whose seeds unfortunately failed togerminate (Muthukumar et al., 1996). Hence, there was no evidence of stable transformation. Similarly, thework of Sahoo et al. (2000) only produces transgenic shoots but did not provide evidence of stable integration.Furthermore, Ikea (2003) using an entirely different method of DNA delivery known as particle bombardmentfor cowpea transformation, observed transformation in cowpea. However, further molecular evidence of stabletransformation with Mendelian transmission of the transgenes to progeny was not provided.Recent Advances in Cowpea Transformation:Cracking the hard nut of cowpea transformation is critical to the maximal deployment of recombinant DNAtechnology for genetic improvement of this very important crop of the poor.  Popelka et al. (2006) is the firstreport on cowpea transformation where transgenic cowpeas obey the Mendelian rule of transmitting thetransgenes to their progeny. The workers have really used brute force to ensure stable integration of thetransgene. They used a hypervirulent Agrobacterium strain, AGL1 that habours a binary vector pBSF16 thatcontains a reporter gene (uidA), encoding the â-glucuronidase (GUS) and a selectable marker gene (bar), whichencodes phosphinothricin acetyl transferase (PAT) – a highly reliable selectable marker. Furthermore, the co-cultivation was extended to 6 days, coupled with the addition of thiol-compounds during infection and co-cultivation with Agrobacterium . Additionally, they cautiously employed a lengthy regeneration procedure thatensured there were no escapes from the selection regime, but which took between 5 to 8 months from explants1 preparation to harvest of T seeds. Slightly improved transformation efficiency from 0.05% to 0.15% (Popelkaet al., 2006) to 0.76% was reported by Chaudhury et al. (2007).They used cotelydonary nodal explants asPopelka’s group but they inflicted wounds on the nodal cells by stabbing with sterile needle prior toAgrobacterium  infection.  Additionally, they introduced a second selection regime at the rooting stage, theprocedure that has been found to be critical in the transformation of other related species (Saini and Jawail,2005). Employing the biolistic method of gene transfer, Ivo et al. 2008, reported 0.9% transformation efficiencyfrom bombarded embryonic axes, thereby presenting the first work on the use of this approach for generatingtransgenic cowpea plants that obey the Mendelian law. Yet, much higher transformation efficiencies from1.64% to 1.67% have been reported recently by Sahoo’s group in India.  This remarkable increase in thetransformation efficiency was attributed to the constitutive expression of additional virulence genes in theLBA4404 Agrobacterium strain coupled to a regimen of geneticin selection at 45 mg/l (Solleti et al. 2008a),a more effective selective agent than phosphinothricin and kanamycin that were used by Popelka et al. 2006and Chaudhury et al. 2007, respectively. The group also reported heterologous expression of the common beaná-amylase inhibitor 1 gene in the transgenic seeds (Solleti et al. 2008b), which is probably the first report onthe expression of gene that confers desirable agronomical trait on the cowpea plant. These recent successesin cowpea genetic transformation have therefore pave way for the introduction of more agronomic traits tocowpea, thereby enhancing the genetic diversity of the crop, and consequently complementing existing breedingprogrammes.Implication for Food Security in Developing Countries:The world population is growing at a fast pace, and it is projected to be well over 7 billion by 2020. Itis pathetic to note that the developing world, which is under pressure with the challenge of food insecurity,is contributing most to this increase (Pardey and Wright, 2002). This paints a bleak picture of the future’s foodsecurity, especially in the developing countries of the sub-Saharan Africa; as food production is currently notmatching up with the population growth. With over 200 million Africans depending on cowpea daily, as themajor source of protein and vitamins, common sense suggests that the current production level of the cropshould be increased dramatically in order to prevent a major future crisis.  Attack of insect pests remains themajor constraint to cowpea production and storage. The recent advances in cowpea transformation have sparkednew hope that the menace of insect pests can be dramatically reduced by introducing more resistance genesinto cowpea.  This would hopefully enhance crop production and seed quality in storage, and consequentlyprovide higher incomes for the poor farmers.  1084Aust. J. Basic & Appl. Sci., 3(2): 1083-1086, 2009Directions:Since a good number of insect-resistance genes, such as those that encode plant lectins, á-amylaseinhibitors, protease, chitinases, as well as Bacillus thuringiensis crystal proteins are available, an importantimmediate challenge is to aggressively transform these genes into elite cowpea lines. Consideration could alsobe given to gene pyramiding strategies in order to accumulate insect resistance in the crop, as a way to ensureeffective resistance to most of the insect pests. Hence, collaborative efforts should be facilitated amongscientists working in this field, particularly in the concerned regions, to generate tailor-made cowpea plantsthat can now successfully challenge these ravaging pests in their respective agro-ecological zones. REFERENCESAkella, V. and P.F. Lurquin, 1993. Expression in cowpea seedlings of chimeric transgenes afterelectroporation into seed-derived embryos. Plant Cell Reports, 12: 110-117.Chandra, A. and D. Pental, 2003. Regeneration and genetic transformation of grain legumes: An overview.Current Science, 84(3): 381-387.Chaudhury, D., S. Madanpotra, R. Jawail, R. Saini, P.A. Kumar and P.K. Jaiwal, 2007. Agrobacteriumtumefaciens-mediated high frequency genetic transformation of an Indian cowpea (Vigna unguiculata L.)cultivar and transmission of transgenes into progeny. Plant Science, 172: 692-700.Filippone, E. and R. Penza, 1992. Agrobacterium tumefaciens-mediated gene transfer. In: Thottappilly G,Monti LM, Mohn Raj DR, Moore AW (eds) Biotechnology: enhancing research on tropical crops in Africa.CTA/IITA copublication, IITA, Ibadan, pp: 197-202.Garcia, J.A., J. Hillie and R. Goldbach, 1986. Transformation of cowpea (Vigna unguiculata) cells withan antibiotic resistance gene using Ti-plasmid derived vectors. Plant Science, 44: 37-46.Garcia, J.A., J. Hillie and R. Goldbach, 1986. Transformation of cowpea (Vigna unguiculata)cells with afull length DNA copy of cowpea mosaic virus mRNA. 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Exciting Times for Cowpea Genetic Transformation Research

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Exciting Times for Cowpea Genetic Transformation Research

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