REVIEW OF LITERATURE
2.2. Regeneration and genetic transformation of cowpea
active form of αAI-Pa1 in transgenic azuki bean was a single polypeptide with a size similar to that expected for the full-length encoded protein. These results suggested that αAI-Pa1 defining a new type of bean αAI, structurally related to lectins and not activated by proteolytic processing.
other than young leaves for somatic embryos induction in solid and liquid suspension cultures need thorough investigation. The basal medium developed for embryo development by Pellegrineschi et al. (1997) could form a starting point for formulating media for growth of somatic embryos in vitro. Growth medium supplements that enhanced embryo development included addition of sucrose, casein hydrolysate, and any one of three commonly used cytokins, namely zeatin, benzyl amino purine (BAP), and kinetin, for enhancing embryo maturation. Prem Anand et al. (2000) stressed the importance of organic supplements along with 2,4-D for the induction of embryogenic calli. Ramakrishnan et al. (2005a) reported an efficient protocol for plantlet regeneration from the cell suspension cultures from primary leaf-derived embryogenic calli of cowpea. The sequence of events in the functional body pattern formation during the somatic embryo development was described in cowpea suspension cultures (Ramakrishnan et al. 2005b).
The establishment and maintenance of embryogenic cultures as well as recovery of plants can be an extremely labor intensive and lengthy process that has the added risk of encountering morphological abnormalities and sterility among regenerants. In contrast, multiple shoot formation via organogenesis is simpler once a suitable explant has been identified. Various laboratories have independently reported successful regeneration of cowpea by direct organogenesis from a variety of explants. Kartha et al. (1981) described a method for cowpea regeneration from shoot apical meristem at low level of exogenously supplied cytokinin. The authors attributed the presence endogenous hormones in cowpea meristems to be adequate to effect plant regeneration. Multiple shoot induction from mature de-embryonated cotyledons with intact proximal end was reported albeit with low frequency in cowpea (Muthukumar et al. 1996). Addition of putrescine to high cytokinin medium enhanced the shoot forming response of axenic hypocotyls and cotyledons excised from green immature pods in five different genotypes of cowpea (Pellegrineschi, 1997). Fertile cowpea plants have been regenerated successfully using nodal thin cell layer (TCL) explants (Le et al. 2002). The TCL, approximately eight cells thick, was obtained by cutting twice over each cotyledonary node, followed by regeneration on MS media containing either zeatin and indole butyric acid (IBA) or BAP and IBA. Brar et al. (1999a) reported a regeneration system from cotyledon that was applicable to 17 US commercial cowpea cultivars and breeding lines. The regeneration capacity was reported to be significantly influenced by the BA concentration in the initiation stage, culture duration, and genotype used. The inclusion of silver nitrate, an ethylene inhibitor in shoot induction medium has been shown to improve regeneration from cotyledon of cowpea (Brar et al. 1999b). Popelka et al. (2006) reported multiple shoot formation from longitudinally bisected cotyledonary node explants in different BAP, and TDZ containing medium. The addition of TDZ resulted in formation of deformed shoots which could not be
elongated beyond the bud stage on transferring to medium containing low concentration of TDZ. An in vitro regeneration system from shoot apices was reported in cowpea cv.
Blackeye, known for high yield potential as well as early flowering and maturation (Mao et al. 2006). Chaudhury et al. (2007) reported the multiple shoot induction and plant regeneration in Indian cowpea cultivar, V-585 from cotyledonary node explants without cotyledons.
These studies have, however, indicated that cowpea in vitro regeneration responses are genotype dependent requiring the optimization of tissue culture condition for each individual genotype. Furthermore, regeneration response was dependent on the type of explant used (Brar et al. 1999a), basal salt compositions, plant growth regulators (PGRs) and sucrose levels (Pellegrineschi, 1997; Popelka et al. 2006). Since nutritional requirements of different explants are different, a systematic optimization of media components and culture conditions for each individual explant type may overcome variable response in in vitro regeneration (Pellegrineschi, 1997; Popelka et al. 2006).
2.2.2. Genetic transformation of cowpea
Absence of efficient and routine genetic transformation system in cowpea poses a formidable challenge in the incorporation of candidate genes for biotic and abiotic stress tolerance.
Although genetic transformation of cowpea has been difficult and challenging till date, considerable progress has been made in regeneration of stable transgenic plants through both Agrobacterium as well as biolistic mediated method (Solleti et al. 2008).
Garcia et al. (1986, 1987) were the first to investigate whether cowpea was susceptible to Agrobacterium tumefaciens infection and demonstrated that foreign genes could be stably introduced and expressed in cowpea cells. The authors recovered kanamycin-resistant calli from leaf discs of cowpea on infection with Agrobacterium (Garcia et al. 1986) and subsequently reported introduction of a full-length cDNA copy of the of cowpea mosaic virus, under the control of the 35S CaMV promoter or the nopaline synthase promoter into leaf- discs in order to study the interaction between cowpea mosaic virus and the natural host (Garcia et al. 1987). Northern blot analysis revealed the expression of the transgene in transformed calli confirming its stable integration. However, no plants could be regenerated from kanamycin-resistant calli. Stably transformed calli of cowpea was recovered from mature embryos, cotyledonary node buds, epicotyls, and apical meristems upon cocultivation with A. tumefaciens (Perkins et al. 1987; Filippone, 1990) using hypervirulent strain 6044.
Penza et al. (1991) used sliced mature cowpea embryos as a target for genetic transformation by A. tumefaciens and were able to regenerate putatively transgenic plants. However, genetic evidence of transgene integration was not presented. Transgenic hairy roots were produced
from leaf, epicotyl, and hypocotyl explants of cowpea on co-cultivation with A. rhizogenes harbouring soybean cell wall protein gene (SbPRP1) promoter-GUS construct (Suzuki et al.
1993). The authors showed localization of SbPRP1 in actively growing roots (apical and elongating regions) during seedling growth. Nagl and Ehemann (1994) reported the transient and stable GUS expression in the calli obtained from transformed protoplasts of cowpea ssp.
susquipedalis.
The first production of transgenic cowpea plants was reported by Muthukumar et al. (1996).
These authors generated hygromycin resistant primary transformed shoots from mature de- embryonated cotyledons cocultivated with A. tumefaciens and confirmed the stable integration of transgene. The hygromycin-resistant shoots grew to maturity and set seed.
Nevertheless, none of the seeds germinated and no evidence of transgene transmission to the progeny was obtained. The group suggested further studies to produce viable seeds for analysis of inheritance of the transgene. Kononowicz et al. (1997) reported the production of chimeric plants of cowpea from shoot apices of young seedlings as well as from embryonic axis and cotyledonary segments of immature seeds, under kanamycin selection, using both A.
tumefaciens and particle bombardment method for gene transfer. Sahoo et al. (2000) reported the recovery of chimeric transformed plants of cowpea under kanamycin selection through Agrobacterium-mediated transformation of shoot apices. An extra wounding treatment in shoot apices prior to cocultivation was found necessary for optimal gene delivery to shoot apices. Popelka et al. (2006) obtained transgenic plants of cowpea through Agrobacterium- mediated transformation of longitudinally bisected embryonic axes attached to the cotyledons.
Addition of thiol compounds during infection and co-culture, prolonged co-culture, application of a delayed selection in the protocol resulted in recovery of transgenics with a frequency of transformation of 0.15%. Transgenic lines transmitted the transgene (bar gene) to their progenies following Mendelian inheritance (Popelka et al. 2006). Chaudhury et al.
(2007) used cotyledonary node explants to generate fertile transgenic plants with an efficiency of 0.76%. Transgenic plants were recovered on kanamycin selection and they inherited the transgenes in Mendelian fashion in the first generation. However, both the transformation systems (Popelka et al. 2006; Chaudhury et al. 2007) are time consuming and present low frequency of germ line transformation. Solleti et al. (2008) reported a dramatic increase in efficiency of T-DNA delivery to cotyledonary node explants of cowpea by constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404.
The authors employed a geneticin based selection system for rapid and efficient identification of transgenic shoots, and synergistic use of BAP and kinetin for optimal multiplication and elongation of transformed shoots. Stable transgenic plants transmitting transgenes to
progenies were obtained with a frequency of 1.64% which was significantly higher than previous reports on cowpea transformation.
The transient expression of reporter genes in cowpea seedlings following the electroporation of zygotic embryos with plasmid DNA harbouring the chimeric gus gene has been demonstrated (Penza et al. 1992; Akella and Lurquin, 1993). Delivery of linearised DNA following electroporation of seedling tissues was demonstrated in cowpea with remarkably high transient GUS expression in intact hypocotyls and epicotyls (Dillen et al. 1995).
However, no stably transformed cowpea plants have been obtained through electroporation mediated transformation. Ikea et al. (2003) were able to generate transgenic cowpea plants after particle bombardment of embryonic axes. However, the transgenes were transmitted to only a small proportion of the progeny and no evidence for stable integration was presented.
The authors presumed an unstable transgene integration, which could not be fixed in a homozygous line. In addition, the tissue culture protocol used was time consuming, involving several treatments and medium transfers of the bombarded embryos, prior to achieving putative transgenic plantlets. Ivo et al. (2008) demonstrated a novel system of exploiting the biolistic process to generate stable transgenic cowpea plants by combining the use of the herbicide imazapyr to select transformed meristematic cells after physical introduction of the mutated ahas gene (coding for a mutated acetohydroxyacid synthase, under control of the ahas 5’ regulatory sequence) and a simple regeneration system from shoot apices. The authors generated transgenic plants with a frequency of 0.90%. The progenies (first and second generations) of all self-fertilized transgenic lines revealed the presence of the transgenes (gus and ahas) cosegregated in a Mendelian fashion (Ivo et al. 2008).