Use of ex vitro composite plants to study the interaction of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides
© Mellor et al.; licensee BioMed Central Ltd. 2012
Received: 16 May 2012
Accepted: 21 June 2012
Published: 28 June 2012
Cowpea (Vigna unguiculata L.) is an important grain and forage legume grown throughout sub-Saharan Africa primarily by subsistence farmers on poor, drought prone soils. Genetic improvement of the crop is being actively pursued and numerous functional genomics studies are underway aimed at characterizing gene controlling key agronomic characteristics for disease and pest resistances. Unfortunately, similar to other legumes, efficient plant transformation technology is a rate-limiting step in analysis of gene function in cowpea.
Here we describe an optimized protocol for the rapid generation of transformed hairy roots on ex vitro composite plants of cowpea using Agrobacterium rhizogenes. We further demonstrate the applicability of cowpea composite plants to study gene expression involved in the resistance response of the plant roots to attack by the root parasitic weed, Striga gesnerioides. The utility of the new system and critical parameters of the method are described and discussed herein.
Cowpea composite plants offer a rapid alternative to methods requiring stable transformation and whole plant regeneration for studying gene expression in resistance or susceptibility responses to parasitic weeds. Their use can likely be readily adapted to look at the effects of both ectopic gene overexpression as well as gene knockdown of root associated defense responses and to the study of a broader range of root associated physiological and aphysiological processes including root growth and differentiation as well as interactions with other root pests, parasites, and symbionts.
KeywordsAgrobacterium rhizogenes Composite plants Hairy roots Resistance Striga gesnerioides Witchweed
Cowpea (Vigna unguiculata L. Walp.) is the most important grain legume grown in sub-Saharan Africa [1, 2]. Approximately 12.5 million tons of cowpea grains are produced worldwide each year with a majority (over 64%) of the production taking place on low-input, subsistence farms in West and Central Africa . Two characteristics contribute to its agronomic and economic importance. The plant is generally drought tolerant and can provide some yield even under harsh conditions, and it fixes nitrogen symbiotically thereby enhancing soil fertility especially when used in rotation with cereals [4, 5]. Throughout the Sahel cowpea is referred to as “poor man’s meat” because of its high protein content (20-25%) and good nutritional value . The pods and seeds are consumed at all stages of growth (e.g., green pods, fresh or dry seeds) and the young leaves are often used for soups and stews . In addition to its value as human food, cowpea hay is an important source of animal fodder . Improvement of cowpea as a multifunctional crop is a key breeding concern and significant efforts are currently aimed at its genetic improvement .
Like most crops, cowpea growth and grain yields are greatly reduced by a variety of biotic pests (e.g., bacterial, fungal, and viral diseases, insects, nematodes, and herbivores) and abiotic stresses (severe drought, salinity, and heat) . Among the major biotic constraints is parasitism by Striga gesnerioides (L.) Walp. (Orobanchaceae) commonly referred to as witchweed. Witchweeds are noxious and persistent pests in farmers’ fields and yield losses due to S. gesnerioides parasitism are extensive in the Sudano-Sahelian belt of West and Central Africa . Control of the parasite is difficult because it produces thousands of seeds per generation that remain in the seed bank for years and most of the damage to its host plant occurs prior to its emergence from the ground . The damaging effects of Striga in this region are further compounded by poor soils and drought .
While most cowpea plants are susceptible to Striga parasitism, some local landraces and wild accessions have been identified that are resistant to the parasite, and in most reports resistance is a dominant characteristic, inherited in a monogenic manner [2, 12]. Complicating the identification of Striga-resistant germplasm is the variable nature of the parasite with at least seven distinct races of S. gesnerioides (designated SG1 (Burkina Faso), SG2 (Mali), SG3 (Nigeria and Niger), SG4 (Benin), SG4z (Zakpota region of Benin), SG5 (Cameroon), and SG6 (Sénégal)) now identified throughout West Africa [13–15]. Analysis of several advanced populations segregating for resistance to one or more of the different races of S. gesnerioides has resulted in the genetic mapping of several race-specific resistance (R) genes within the cowpea genome and the development of molecular markers liked to these genes . Using a positional cloning approach, Li and Timko  isolated and characterized a gene (designated RSG3-301) capable of conferring resistance to S. gesnerioides race 3 (SG3). RSG3-301 encodes an R protein homolog containing a coiled-coil (CC) protein-protein interaction domain at the N-terminus, a nucleotide binding site (NBS), and a leucine-rich repeat domain at the C-terminus. Silencing of RSG3-301 in the resistant cultivar B301 leads to susceptibility to race SG3, but does not affect resistance to other races of the parasite, underscoring the specificity of the resistance response .
Resistant cowpea genotypes exhibit two different response mechanisms to Striga attack. When challenged by a known race, cultivars carrying the appropriate race specific resistance gene exhibit a rapid and robust hypersensitive response typified by a browning and necrosis at the site of parasite attachment, and subsequent rapid death of the parasite within 3–4 days [15, 18, 19]. In host plants lacking the appropriate resistance gene, the parasite rapidly penetrates the host root cortex, forms connections to the host vascular system, swells to form a tubercle, and expands its cotyledons leading to subsequent above ground growth and flowering.
Like most legumes, the genetic transformation and regeneration of cowpea has proven to be difficult and challenging . Cowpea is susceptible to genetic transformation by Agrobacterium, and a number of reports have appeared in the literature describing stably integrated and heritable transgenes following A. tumefaciens-mediated transformation [21–28]. However, in vitro regeneration of shots/seedlings from various seedling explants (including primary leaves , epicotyl , mature cotyledon , cotyledonary node [32, 33] and nodal thin cell layer ) appears to be highly dependent on cultivar genotype with even the most promising genotypes giving very low regeneration frequency and numbers of regenerants. Coupled to transformation, the efficiency of recovery of fertile transgenic cowpea plants ranges from 0.1% to a 1-2% [25–28]. Given this limitation, traditional transgenic approaches for gene functional characterization in cowpea are difficult.
Ex vitro composite plants consist of a wild-type shoot with transgenic roots induced by transformation with A. rhizogenes[35–37]. The use of ex vitro composite plants has proven to be a successful alternative strategy for candidate gene analysis and genetic pathway dissection in plants where transformation and regeneration is difficult, time consuming and infrequent. The technique has been applied to the study of root nutrient uptake, hormone transport, interactions with root nodulating bacteria and mycorrhizal symbiotic, and parasitic nematodes [35, 36, 38]. Here, we present a protocol for the rapid development of transformed hairy roots on composite plants of cowpea using A. rhizogenes induced root formation. We further demonstrate the applicability of this approach to studying gene function involved in the interaction of cowpea with the root parasitic angiosperm, S. gesnerioides.
An optimized protocol for the generation of ex vitro composite cowpea plants
At multiple points in the protocol we empirically determined the optimal conditions for recovery of composite cowpea plants. Since composite plants potentially generate a mixture of transgenic and non-transgenic roots, in order to optimize transformation efficiency (defined as the percentage of plants exhibiting one or more transgenic roots) and increasing the portion of transgenic versus untransformed roots, we used high level constitutive expression of green-fluorescent protein (GFP) as a biomarker for transformation. In these studies, plasmid pCambia1300-Gmubi3 , containing the soybean polyubiquitin 3 promoter fused to the coding region of GFP, was introduced into A. rhizogenes R1000 strain and used to determine the best conditions for cowpea transformation.
Among the factors that significantly enhance transformation efficiency, we found that the temperature at which co-cultivation with A. rhizogenes was carried out and the length of the dehydration stress period imposed following co-cultivation were most important. As shown in Figure 3 plants grown at 21°C showed the highest transformation rate. Over 80% of plants infected with Agrobacterium harboring pCambia1300-Gmubi3 binary vector underwent a stable transformation (Figure 3C). Plant transformation efficiency was significantly lower when co-cultivated with A. rhizogenes at 23°C. Less than 40% of plants expressed GFP in their roots. Temperature also had an impact on the percentage of transgenic versus non-transgenic roots formed on the composite plants, with plants transformed at 21°C exhibiting a significantly higher percentage of stably transformed roots (~25% of total roots generated) compared with 23°C (~10% transgenic) and 25°C (5% transgenic), respectively (Figure 3D).
Use of composite plants to study striga-host interactions
We have previously demonstrated that resistance in cowpea to parasitism by S. gesnerioides is conferred by monogenically inherited resistance genes that function in a race-specific manner [16, 17]. The cowpea cultivar Blackeye is known to be susceptible to all races of S. gesnerioides identified in West Africa . Having established conditions for the efficient generation of cowpea composite plants, we next explored whether our optimized protocol could be used for the study of root associated activities, in particular the interaction of cowpea cultivars with S. gesnerioides. To this end the cowpea RSG3-301 gene, known to confer resistance to S. gesnerioides race SG3, was placed under the control of the constitutive CaMV 35S promoter in plasmid pK7WG2 and pK7WG2D yielding pKM0-RSG3-301 and pKMG-RSG3-301, respectively. pKM0-RSG3-301 allows for the overexpression of RSG3-301 alone, whereas pKMG-RSG3-301 expresses both RSG3-301 and GFP. The two plasmids were independently introduced into A. rhizogenes R1000 and used to generate composite plants using the optimized protocol described above. Composite plants were then used to determine whether (i) transformed roots of composite cowpea plants responded similarly to wild-type roots with respect to the resistance/susceptibility phenotype to attempted parasitism by S. gesnerioides, and (ii) whether introduction of a race-specific resistance gene (RSG3-301) into a susceptible cowpea genetic background (i.e., the Blackeye cultivar) confers race-specific resistance demonstrable in composite plants roots.
Tabular data of relative RSG3-301 transcript levels in transgenic and non-transgenic roots of cowpea composite plants
Average relative transcript levels
Discussion and conclusions
The recalcitrance of cowpea to efficient transformation and regeneration has been a bottleneck in functional genomics analysis and bioengineering for genetic improvement of the crop. The current study addresses one of these fundamental limitations by providing a fast and relatively easy method to rapidly generate and evaluate multiple independent transformants for screening gene expression associated with root localized plant functions. The transformation efficiency we have achieve (up to 80% plants with at least one transgenic root; and 25% of all roots formed being transgenic) with our optimized protocol is comparable to that obtained by Collier et al  for soybean who reported between 2 and 4 transformed roots per transformed plant with a transformation efficiency of ~ 80%. It is also similar to that reported for Medicago Phaseolus and pea [35, 37, 38] although direct comparisons are more difficult because of the manner in which transformation rates were measured. Nonetheless, the recovery of transgenic events we achieve are significantly higher than the efficiency of recovery of fertile transgenic cowpea plants using traditional A. tumefaciens –based transformation and regeneration which range from 0.1% to a 1-2% [25–28]. It should be noted that the limitations of the composite system are that although these are stable transformations they do not allow recovery of stable fertile offspring and therefore analysis is constrained to only the initial generation.
The great advantage of the composite system is the utility for studying root associated parameters such as parasitism by weedy parasitic angiosperm and other root pests and pathogens. In this regard, the use of composite plants make it possible to look at the effects of both ectopic gene overexpression as well as gene knockdown on root associated defense responses. As demonstrated in our above studies, the composite plant system was capable of recapitulating what is found in non-transgenic plants with regard to the resistance or susceptibility response to attempted Striga parasitism. Overexpression of a race-specific resistance gene (e.g., RSG3-301) in a susceptible genotype resulted in acquisition of the resistance phenotype similar to what is observed in a naturally occurring resistant cultivar such as B301. This observation indicates that the process of transformation does not fundamentally change the nature of the signal transduction apparatus involved in defense in the transgenic roots. This might have been the case since the introduction of ROL genes by A. rhizogenes and generation of the hairy-root phenotype is known to be associated with alterations in phytohormonal concentrations . Clearly, this is not significantly impacting the defense processes (or lack thereof) in the plant. This result opens up the potential for using the composite plant approach to evaluate various candidate genes that might be involved in cowpea resistance or susceptibility to S. gesnerioides, as well as other root parasitic angiosperms of economic significance such as Alectra vogelii. The utility as noted by Collier et al  lies in the fact that candidate gene constructs identified by screening in composite plants can be moved immediately without further manipulation to A. tumefaciens for the production of stable transgenic plants. In the case of cowpea, where the efficiency of traditional A. tumefaciens –based transformation and regeneration is low, this would result in a significant economy of time and effort.
The cowpea composite plant system also has applicability to studying gene expression in other physiological processes such as root development, growth and differentiation, and aphysiological conditions such as bacterial, fungal or viral induced diseases and interactions of roots with parasitic nematodes, nodulation by symbiotic bacteria, and colonization by mychorrizal fungi.
Materials and methods
Plant materials and growth
Descriptions of the cowpea accession used in this study can be found in . B301 is a Striga multi-race resistant cultivar from Botswana; Blackeye is a Striga susceptible commercial variety. Seeds of the cowpea cultivars B301 and Blackeye were surface-sterilized with 1% Metricide (Metrex, Romulus, MI) for 5 min . They were then placed between two sheets of moist glass fiber filter paper (GF/A Whatman; Piscataway, NJ), held between two blocks of moistened rockwool (Grodan Inc., Milton, ON) for 10 days until leaves have fully developed. Plants were grown at 30°C in a growth chamber to ensure moist environment. The chamber was covered with a transparent dome which light passage.
Vector construction and generation of Agrobacterium rhizogenes strains
The structure of the pCambia1300-Gmubi3 vector has been previously reported  and contains green fluorescent protein (GFP) coding region under the control of the soybean polyubiquitin 3 promoter. Gateway compatible destination vectors pK7WG2 and pK7WG2D  were obtained commercially (VIB, Brussels, Belgium). For overexpression of the cowpea RSG3-301 resistance gene , the RSG3-301 coding region was mobilized into the pENTR/D-TOPO intermediate vector (Invitrogen, Grand Island, NY) and pENTR/D-TOPO-RSG3-301 recombined with pK7WG2D using the LR recombination reaction in the Gateway LR Clonase Enzyme mix (Invitrogen, Grand Island, NY). The resulting plasmid is designated, pKMG-RSG3-301. The RSG3-301 coding region in pENTR/D-TOPO-RSG3-301 was similarly recombined with pK7WG2 generating pKM0-RSG3-301. The relevant portions of pKMG-RSG3-301 and pKM0-RSG3-301 were sequenced to confirm the integrity of the introduced gene.
The various plasmids (pCambia1300-Gmubi3, pKMG-RSG3-301 and pKM0-RSG3-301) were transformed into Agrobacterium rhizogenes R1000 competent cells by electroporation and transformed colonies were selected by growth for two days on solid YEB medium containing 0.1 mM KAN. Independent positive transformants were picked and inoculated into liquid YEB medium containing 0.1 mM KAN and grown at 28°C until saturation. Cells were collected and plasmid DNA was prepared using the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Glen Burnie, MD). The presence of the introduced plasmid into the Ti plasmid was verified by restriction digestion using BsrG1 enzyme (New England Biolabs, Ipswitch, MI). Colonies of Agrobacteria confirmed to be transformed with the various constructs were selected, grown in liquid culture, and stored at −80°C in 50% glycerol solution until ready to use.
Two days prior to plant transformation, A. rhizogenes carrying pKMG-RSG3-301, pKM0-RSG-301, pK7WG2D and pK7WG2 plasmids were thawed and streaked out on solid YEB medium containing 0.1 mM (50 mg/l) KAN, 0.4 mM acetosyringone and grown at 28°C. Individual colonies were picked and inoculated into 10 ml liquid YEB media with antibiotic and supplement as mentioned above and grown overnight until OD600 = 0.8 was reached (OD600 of 1.0 = 5 x 108 cells/ml).
On the day of plant transformation, bacterial suspension was spun down at 3,500 rpm and resuspended in 15 ml 20% MS media with vitamins (Caisson labs, Logan, UT) supplemented with 0.4 mM acetosyringone. Bacteria were grown for another several hours until OD600 = 0.8 was reached and then 6 ml of the bacterial suspension was pipetted onto a 1 cm3 rockwool cube (Grodan Inc., Milton, ON) so that each cube was saturated.
Ten day-old cowpea plants were taken out of growth chamber, and the roots excised approximately 3 cm below the cotyledons using a sterile razor blade. The plant was then inserted into the bacteria-saturated rockwool cube and placed into a darkened growth chamber at 21°C to ensure high transformation efficiency. On the second day, a lid from the growth chamber was removed which allowed plants to dry. Applying drought stress appeared a crucial factor for increasing amount of transformed roots. Plants were rewatered after leaves became obviously wilted. This procedure was repeated two days in the row. On the fourth day plants were transferred into a 22°C growth room and allowed to grow in cubes. First transformed roots emerged from the cube 12–14 days after transformation. Plants were then taken from growth room and rockwool surrounding emerging roots was gently removed using forceps. Cowpea was then transferred to 24 cm x 24 cm x 3 cm growth chamber containing rockwool with a 100 μm mesh separating the cowpea roots from the rockwool . Cowpea seedlings were grown for another 7 days in a controlled environment growth room under a 12 H light–dark photoperiod at 30°C. After a week, plants infected with pKMG-RSG3-301 were tested for GFP present in their roots using Zeiss Stereo Discovery V20 with Axciocam MRc High resolution camera. Several roots (transformed and non-transformed) were then collected and stored in −80°C for qRT-PCR to assess RSG3-301 transcript levels in the tissue. Due to the lack of GFP, plants infected with pKM0-RSG3-301 were only collected for qRT-PCR.
Assessing Striga – host interactions
S. gesnerioides is a federally regulated noxious weed. All work involving viable S. gesnerioides seeds, developing parasites, and all analysis of host-parasite interactions, were performed in our APHIS approved quarantine facility at UVA (Facility Number 669; Permit No. P526P-11-03310). Seeds of the various S. gesnerioides races were collected in the field as reported previously . Seeds of S. gesnerioides race SG3 were surface sterilized, pre-conditioned for 9 days , and germination triggered using root exudates from cowpea cultivar B301. Pre-germinated seeds (50 mg equivalent to ~7500-10,000 seeds, with an average germination of 75% at time of infestation) were gently transferred on developed cowpea roots (pKMG-RSG3-301 fluorescent and non-fluorescent; pK7WG2D fluorescent and non-fluorescent) using a paint brush. Growth chambers were closed and left for another 8 days for infection to occur. After 8 dpi, infection rate was assessed. Number of events (tubercle formation and hypersensitive response) was counted on plants infected with pKMG-RSG3-301 for both fluorescent and non-fluorescent roots as well as plants infected with pK7WG2D. Number obtained from 14 individual plants per treatment were averaged and presented ± SE.
Verification of RSG3-301 transcript levels using qRT-PCR
qRT-PCR was used to validate RSG3-301 transcript levels of RNA samples prepared from Blackeye root tissues subject to the various treatments described above. Total RNA was extracted as described in  with minor modifications and quantified using a Nanodrop instrument (Thermo Scientific, Wilmington, DE). cDNA was synthesized from 2 μg of DNAse- treated (Roche, Indianapolis, IN) total RNA using an Invitrogen Thermoscript Kit (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. The reaction was carried out in triplicate.
For qRT-PCR reactions, gene-specific primers were designed against the RSG3-301 gene coding region  that give a 147 bp amplicon and an actin reference gene (XM_003521168; GI: 356504867) giving a 132 bp amplicon. The primers used are as follows: RSG3-301-F: 5′- AGTAAGGGATGTTGGAAGCAA -3′and RSG3-301-R: 5′- AATTACATCAGACTCGGGAAT -3′); Actin-F: 5′- CGAGCAGGAATTGGAAAC-3′and Actin-R: 5′-ATCATGGATGGCTGGAAC-3′. Amplification reactions (25 μl) containing 15 ng of cDNA were carried out in Multiplate PCR 96-well plate formats (Bio-Rad, Hercules, CA), using the iQ SYBR Green supermix kit (Bio-Rad, Hercules, CA) in an iCycler Optical Module PCR instrument according to the manufacturer’s instructions quantification of relative transcript abundance was performed described by Schmittgen and Livak  by calibrating threshold cycles of the RSG3-301 transcripts with that of the actin reference gene. The equation 2(−ΔΔCq) was used to calculated relative expression (difference in concentration between samples based on normalization with a reference gene), in which Cq is the cycle number of the threshold point at which the fluorescence is detectable. A total of 5 independent biological replicates were used and mean and standard error (SE) were determined.
days post- inoculation
Green fluorescent protein
Murashige and skoog
quantitative reverse transcriptase polymerase chain reaction
Revolutions per minute
Yeast extract and beef.
We thank Kan Huang for helpful comments on various aspects of this work. This work was supported in part by grants from the Kirkhouse Trust and Natural Science Foundation (IBN-0322420 and IOS-07-01748; CR-20006-477823) awarded to MPT.
- Ehlers JD, Hall AE: Cowpea (Vigna unguiculata L. Walp). Field Crop Res. 1997, 53: 187-204. 10.1016/S0378-4290(97)00031-2.View ArticleGoogle Scholar
- Timko MP, Singh BB: Cowpea, a Multifunctional Legume. Genomics of Tropical Crop Plants. Edited by: Moore PH, Ming R. 2008, Springer Science + Business Media, LLC, New York, 227-258.View ArticleGoogle Scholar
- Langyintuo AS, Lowenberg-DeBoer J, Faye M, Lambert D, Ibro G, Moussa B, Kergna A, Kushwaha S, Musa S, Ntoukam G: Cowpea supply and demand in West and Central Africa. Field Crop Res. 2003, 82: 215-231. 10.1016/S0378-4290(03)00039-X.View ArticleGoogle Scholar
- Elowad HOA, Hall AE: Influences of early and late nitrogen fertilization on yield and nitrogen fixation of cowpea under well-watered and dry field conditions. Field Crop Res. 1987, 15: 229-244. 10.1016/0378-4290(87)90012-8.View ArticleGoogle Scholar
- Sanginga N, Dashiell KE, Diels J, Vanlauwe B, Lyasse O, Carsky RJ, Tarawali S, Asafo-Adjei B, Menkir A, Schulz S, Singh BB, Chikoye D, Keatinge D, Ortiz R: Sustainable resource management coupled to resilient germplasm to provide new intensive cereal-grain-legume-livestock systems in the dry savanna. Agr Ecosyst Environ. 2003, 100: 305-314. 10.1016/S0167-8809(03)00188-9.View ArticleGoogle Scholar
- Diouf D, Hilu K: Microsatellites and RAPD markers to study genetic relationships among cowpea breeding lines and local varieties in Senegal. Genet Resour Crop Ev. 2005, 52: 1057-1067. 10.1007/s10722-004-6107-z.View ArticleGoogle Scholar
- Quaye W, Adofo K, Madode Y, Abdul-Razak A: Exploratory and multidisciplinary survey of the cowpea network in the Tolon-Kumbungu district of Ghana: A food sovereignty perspective. Afr J Agric Res. 2009, 4: 311-320.Google Scholar
- Tarawali SA, Singh BB, Gupta SC, Tabo R, Harris F, Nokoe S, Fernández-Rivera S, Bationo A, Manyong VM, Makinde K, Odion EC: Cowpea as a key factor for a new approach to intergrated crop-livestock system research in the dry savannas of West Africa. Challenges and opportunities for enhancing sustainable cowpea production. Proceedings of the World Cowpea Conference III held at the International Institute of Tropical Agriculture (IITA): 4–8 September 2000. Edited by: Fatokun CA, Tarawali SA, Singh BB, Kormawa PM, Tamò M. 2002, , Ibadan, Nigeria, 233-251.Google Scholar
- Parker C: Observations on the current status of Orobanche and Striga problems worldwide. Pest Manag Sci. 2009, 65: 453-459. 10.1002/ps.1713.View ArticlePubMedGoogle Scholar
- Ejeta G: Breeding for Striga resistance in sorghum: Exploitation of an intricate host-parasite biology. Crop Sci. 2007, 47: S216-S227.View ArticleGoogle Scholar
- Obilana AT: Breeding cowpeas for Striga resistance. Parasitic Weeds in Agriculture. Edited by: Musselman LJ. 1987, CRC Press, Boca Raton, 243-253.Google Scholar
- Ejeta G, Gressel J: Integrating New Technologies for Striga Control: Towards Ending the Witch-Hunt. 2007, World Scientific, Hackensack, NJView ArticleGoogle Scholar
- Lane JA, Child DV, Reiss GC, Entcheva V, Bailey JA: Crop resistance to parasitic plants. The Gene-for-Gene Relationship in Plant-Parasite Interactions. Edited by: Crute IR, Holub EB, Burdon JJ. 1997, CAB, Wallingford, UK, 81-87.Google Scholar
- Lane JA, Moore THM, Child DV, Bailey JA: Variation in the virulence of Striga gesnerioides on cowpea: New sources of resistance. Advances in Cowpea Research. Edited by: Singh BB, Mohan Raj DR, Dashiell KE, Jackai LEN. 1997, International Institute for Tropical Agriculture and Japan Agricultural Research Centre for Agricultural Sciences, Ibadan, Nigeria, 225-230.Google Scholar
- Botanga CJ, Timko MP: Phenetic relationships among different races of Striga gesnerioides (Willd.) Vatke from West Africa. Genome. 2006, 49: 1351-1365. 10.1139/g06-086.View ArticlePubMedGoogle Scholar
- Li J, Lis KE, Timko MP: Molecular genetics of race-specific resistance of cowpea to Striga gesnerioides (Willd.). Pest Manag Sci. 2009, 65: 520-527. 10.1002/ps.1722.View ArticlePubMedGoogle Scholar
- Li J, Timko MP: Gene-for-gene resistance in Striga-cowpea associations. Science. 2009, 325: 1094-1094. 10.1126/science.1174754.View ArticlePubMedGoogle Scholar
- Botanga CJ, Timko MP: Genetic structure and analysis of host and nonhost interactions of Striga gesnerioides (witchweed) from central Florida. Phytopathology. 2005, 95: 1166-1173. 10.1094/PHYTO-95-1166.View ArticlePubMedGoogle Scholar
- Timko MP, Rushton PJ, Laudeman TW, Bokowiec MT, Chipumuro E, Cheung F, Town CD, Chen X: Sequencing and analysis of the gene-rich space of cowpea. BMC Genomics. 2008, 9: 103-10.1186/1471-2164-9-103.PubMed CentralView ArticlePubMedGoogle Scholar
- Somers DA, Samac DA, Olhoft PM: Recent advances in legume transformation. Plant Physiol. 2003, 131: 892-899. 10.1104/pp.102.017681.PubMed CentralView ArticlePubMedGoogle Scholar
- Garcia JA, Hille J, Goldbach R: Transformation of cowpea Vigna unguiculata cells with an antibiotic-resistance gene using a Ti-plasmid-derived vector. Plant Sci. 1986, 44: 37-46. 10.1016/0168-9452(86)90166-4.View ArticleGoogle Scholar
- Garcia JA, Hille J, Vos P, Goldbach R: Transformation of cowpea Vigna unguiculata with a full-length DNA copy of cowpea mosaic virus M-RNA. Plant Sci. 1987, 48: 89-98. 10.1016/0168-9452(87)90135-X.View ArticleGoogle Scholar
- Filippone E, Ng NQ, Monti LM: Genetic transformation of pea (Pisum sativum L.) and cowpea (Vigna unguiculata L.) by co-cultivation of tissues with Agrobacterium tumefaciens carrying binary vectors. Cowpea Genetic Resources. Edited by: Ng NQ, Monti LM. 1990, International Institute for Tropical Agriculture, Ibadan, Nigeria, 175-181.Google Scholar
- Penza R, Lurquin PF, Filippone E: Gene transfer by cocultivation of mature embryos with Agrobacterium tumefaciens: Application to cowpea (Vigna unguiculata Walp.). J Plant Physiol. 1991, 138: 39-43. 10.1016/S0176-1617(11)80727-3.View ArticleGoogle Scholar
- Kononowicz AK, Cheah KT, Narasimha ML, Murdock LL, Shade RE, Chrispeels MJ, Fillippone E, Monti LM, Bressan RA, Hasegawa PM: Developing a transformation system for cowpea (Vigna unguiculata L.Walp.). Advances in Cowpea Research. Edited by: Singh BB, Mohan Raj DR, Dashiell KE, Jackai LEN. 1997, International Institute for Tropical Agriculture and Japan Agricultural Research Centre for Agricultural Sciences, Ibadan, Nigeria, 361-371.Google Scholar
- Sahoo L, Sushma , Sugla T, Singh ND, Jaiwal PK: In vitro plant regeneration and recovery of cowpea (Vigna unguiculata) transformants via Agrobacterium-mediated transformation. Plant Cell Biot Mol Biol. 2000, 1: 47-54.Google Scholar
- Muthukumar B, Mariamma M, Veluthambi K, Gnanam A: Genetic transformation of cotyledon explants of cowpea (Vigna unguiculata L Walp) using Agrobacterium tumefaciens. Plant Cell Rep. 1996, 15: 980-985.PubMedGoogle Scholar
- Popelka JC, Gollasch S, Moore A, Molvig L, Higgins TJV: Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny. Plant Cell Rep. 2006, 25: 304-312. 10.1007/s00299-005-0053-x.View ArticlePubMedGoogle Scholar
- Muthukumar B, Mariamma M, Gnanam A: Regeneration of plants from Primary Leaves of Cowpea. Plant Cell Tiss Org. 1995, 42: 153-155. 10.1007/BF00034232.View ArticleGoogle Scholar
- Pellegrineschi A: In vitro plant regeneration via organogenesis of cowpea [Vigna unguiculata (L.) Walp.]. Plant Cell Rep. 1997, 17: 89-95. 10.1007/s002990050358.View ArticleGoogle Scholar
- Brar MS, Al-Khayri JM, Morelock TE, Anderson EJ: Genotypic response of cowpea Vigna unguiculata (L.) to in vitro regeneration from cotyledon explants. In Vitro Cell Dev-Pl. 1999, 35: 8-12.View ArticleGoogle Scholar
- Brar MS, Moore MJ, Al-Khayri JM, Morelock TE, Anderson EJ: Ethylene inhibitors promote in vitro regeneration of cowpea (Vigna Unguiculata L.). In Vitro Cell Dev-Pl. 1999, 35: 222-225. 10.1007/s11627-999-0082-1.View ArticleGoogle Scholar
- Machuka J, Adesoye A, Obembe OO: Regeneration and genetic transformation in cowpea. Challenges and Opportunities for Enhancing Sustainable Cowpea Production. Edited by: Fatokun C, Tarawali CA, Singh BB, Kormawa PM, Tamo M. 2002, International Institute for Tropical Agriculture, Ibadan, Nigeria, 185-192.Google Scholar
- Van Le B, de Carvalho MHC, Zuily-Fodil Y, Thi ATP, Van KTT: Direct whole plant regeneration of cowpea [Vigna unguiculata (L.) Walp] from cotyledonary node thin cell layer explants. J Plant Physiol. 2002, 159: 1255-1258. 10.1078/0176-1617-00789.View ArticleGoogle Scholar
- Boisson-Dernier A, Chabaud M, Garcia F, Becard G, Rosenberg C, Barker DG: Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol Plant Microbe In. 2001, 14: 695-700. 10.1094/MPMI.2001.14.6.695.View ArticleGoogle Scholar
- Collier R, Fuchs B, Walter N, Kevin Lutke W, Taylor CG: Ex vitro composite plants: an inexpensive, rapid method for root biology. Plant J. 2005, 43: 449-457. 10.1111/j.1365-313X.2005.02454.x.View ArticlePubMedGoogle Scholar
- Colpaert N: Composite Phaseolus vulgaris plants with transgenic roots as research tool. Afr J Biotechnol. 2008, 7: 404-408.Google Scholar
- Clemow SR, Clairmont L, Madsen LH, Guinel FC: Reproducible hairy root transformation and spot-inoculation methods to study root symbioses of pea. Plant Methods. 2011, 7: 46-10.1186/1746-4811-7-46.PubMed CentralView ArticlePubMedGoogle Scholar
- Hernandez-Garcia CM, Bouchard RA, Rushton PJ, Jones ML, Chen X, Timko MP, Finer JJ: High level transgenic expression of soybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline. BMC Plant Biol. 2010, 10: 237-10.1186/1471-2229-10-237.PubMed CentralView ArticlePubMedGoogle Scholar
- Veena V, Taylor CG: Agrobacterium rhizogenes: recent developments and promising applications. In Vitro Cell Dev-Pl. 2007, 43: 383-403. 10.1007/s11627-007-9096-8.View ArticleGoogle Scholar
- Mohamed AH, Housley TL, Ejeta G: An in vitro technique for studying specific Striga resistance mechanisms in sorghum. Afr J Agric Res. 2010, 5: 1868-1875.Google Scholar
- Karimi M, Inze D, Depicker A: GATEWAY((TM)) vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 2002, 7: 193-195. 10.1016/S1360-1385(02)02251-3.View ArticlePubMedGoogle Scholar
- Wang X, Tian W, Li Y: Development of an efficient protocol of RNA isolation from recalcitrant tree tissues. Mol Biotechnol. 2008, 38: 57-64. 10.1007/s12033-007-0073-6.View ArticlePubMedGoogle Scholar
- Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative C-T method. Nat Protoc. 2008, 3: 1101-1108. 10.1038/nprot.2008.73.View ArticlePubMedGoogle Scholar
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