Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny

Abstract

Cowpeas are nutritious grains that provide the main source of protein, highly digestible energy and vitamins to some of the world's poorest people. The demand for cowpeas is high but yields remain critically low, largely because of insect pests. Cowpea germplasm contains little or no resistance to major insect pests and a gene technology approach to adding insect protection traits is now a high priority. We have adapted features of several legume and other transformation systems and reproducibly obtained transgenic cowpeas that obey Mendelian rules in transmitting the transgene to their progeny. Critical parameters in this transformation system include the choice of cotyledonary nodes from developing or mature seeds as explants and a tissue culture medium devoid of auxins in the early stages, but including the cytokinin BAP at low levels during shoot initiation and elongation. Addition of thiol-compounds during infection and co-culture with Agrobacterium and the choice of the bar gene for selection with phosphinothricin were also important. Transgenic cowpeas that transmit the transgenes to their progeny can be recovered at a rate of one fertile plant per thousand explants. These results pave the way for the introduction of new traits into cowpea and the first genes to be trialled will include those with potential to protect against insect pests.

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

“…Similar affirmative effects has been reported in several leguminous species such as Pisum sativum (Svabova and Griga 2008), Glycine max (Santarem et al 1998;Ko and Korban 2004), Vigna angularis (Yamada et al 2001), Phaseolus vulgaris (De Clercq et al 2002), Vigna mungo (Saini et al 2003;Muruganantham et al 2007), Vigna unguiculata (Popelka et al 2006) and Vigna radiata (Sonia et al 2007). Acetosyringone increases transformation potential of Agrobacterium strain by enhancing vir functions during transformation (Stachel et al 1986) and has been used in several other plant species also (Atkinson and Gardner 1991;Kaneyashi et al 1994).…”

“…Our results were in accordance with these observations. Higher transformation frequency has been reported at low pH of bacterial inoculation and co-culture media (Popelka et al 2006;Sonia et al 2007;Solleti et al 2008). Inclusion of antioxidants like L-cysteine (5.0 mM) and DTT (1.5 mM) completely inhibited the browning and necrosis of explants cells and increased the number of explants showing intense GUS activity.…”

“…DTT is also an efficient antioxidant with potential anti-necrotic property and is least toxic for plant cells (Svabova and Griga 2008). The beneficial role of thiol compounds on greater survival of explants during co-cultivation has been reported in soybean (Olhoft et al 2001(Olhoft et al , 2003Zeng et al 2004), Vigna unguiculata (Popelka et al 2006), Zea maize (Frame et al 2002) and Vitis vinifera (Das et al 2002a, b).…”

“…In the present study, transformation frequency was highest when co-cultivation was performed at 24°C. Co-cultivation at low temperature has been reported in several instances Kapila et al 1997;Dillen et al 1997;Popelka et al 2006). It has also been found that the temperature dependence profile of T-DNA transfer does not parallel with that of vir gene induction, indicating that another factor possibly the formation of a conjugal pilus is causing temperature sensitivity of T-DNA transfer .…”

Production of herbicide-resistant cowpea (Vigna unguiculata L.) transformed with the bar gene

“…Furthermore, variability is involved in almost every aspect of the regeneration systems explored, such as optimal explant tissues, basal salt compositions, plant growth regulators, sucrose levels, and variation in composition of gelling agents (agar or Gelrite) (Pellegrineschi, 1997;Popelka et al, 2006;Aasim et al, 2009a). Establishment of an efficient and reproducible transformation system based on plant tissue culture using various explants is difficult and highly cultivar-dependent (Muthukumar et al, 1995;Prem Anand et al, 2001;Choi et al, 2003;Chaudhury et al, 2007).…”