Optimization of protocol for multiple shoots regeneration from apical meristem of embryonic axes in cowpea (<i>Vigna unguiculata</i> L. Walp)

Abstract

In this work, we report a new approach for meristem of embryonic axes in four elite cowpea varieties. The hypocotyl and primordial leaves were removed and embryonic axes were placed in culture bottles containing Murashige and Skoog basal + B5 vitamins (MSB5) fortified with 22.2µM, 26.6µM or 31.0µM N 6-benzylaminopurine (BAP obtained on regeneration media consisting of MSB5 supplemented with low concentration (2.22µM) of BAP. Hormone free media promoted direct regeneration of single shoots from the apical meristems of embryonic axes, while the presence of BAP promoted callus induction and indirect regeneration of multiple shoots. Of the BAP concentrations evaluated, produced the highest regeneration frequencies (20.3%, 28.5% and 23.8%) and mean shoots numbers (4.3, 4.0 and 4.1) in SAMPEA 7, SAMPEA 10 and SAMPEA 12 respectively. While, in SAMPEA 9 the highest number of shoots was produced when MSB5 was fortified with 31.0µM BAP. Regenerated plantlets were rooted on hormone free media and acclimatized in the greenhouse. Morphologically normal and fertile progenies were obtained from the greenhouse established generated plants. This protocol could be useful in the genetic engineering of cowpea thereby generating useful transgenic lines for future breeding programmes.

Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants

“…De novo shoot organogenesis using EA as explants A rapid, efficient and reproducible regeneration system is a prerequisite for establishment of an efficient cowpea genetic transformation system. Although several studies of in vitro regeneration of cowpea based on organogenesis have been reported (Aasim et al, 2010;Abdu Sani et al, 2015;Mamadou et al, 2008;Manman et al, 2013;Odutayo et al, 2005;Raveendar et al, 2009;Sani et al, 2018;Tie et al, 2013;Yusuf et al, 2008), an efficient cowpea regeneration system that enables highly efficient transformation is still lacking (Manman et al, 2013). Soybean (Glycine max) transformation based on the pre-existing meristems of EA explants has been well established and provides a reliable and highly efficient means for introducing transgenes (Aragão et al, 2000;Liu et al, 2004;Turlapati et al, 2008).…”

Developing a rapid and highly efficient cowpea regeneration and transformation system using embryonic axis explants

“…De novo shoot organogenesis using EA as explants A rapid, efficient and reproducible regeneration system is a prerequisite for establishment of an efficient cowpea genetic transformation system. Although several studies of in vitro regeneration of cowpea based on organogenesis have been reported (Aasim et al, 2010;Abdu Sani et al, 2015;Mamadou et al, 2008;Manman et al, 2013;Odutayo et al, 2005;Raveendar et al, 2009;Sani et al, 2018;Tie et al, 2013;Yusuf et al, 2008), an efficient cowpea regeneration system that enables highly efficient transformation is still lacking (Manman et al, 2013). Soybean transformation based on the preexisting meristems of EA explants has been well established and provides a reliable and highly efficient mean for introducing transgenes (Aragão et al, 2000;Liu et al, 2004;Turlapati et al, 2008).…”