Biolistic-mediated transient gene expression in shoot apical meristems of the prickly-pear (Opuntia ficus-indica)

Abstract

ABSTRACT

Factors affecting genetic transformation by particle bombardment of the prickly pear cactus (O. ficus-indica)

“…The genetic transformation of nopal is a very difficult process, as has been reported previously (Llamoca-Zarate et al 1998 , 1999a , b , Silos-Espino et al 2006 ; Angulo-Bejarano 2013 ). In particular, genetic transformation by particle bombardment is marked by low efficiency of stably transformed cells, and thus, condition optimization is necessary.…”

“…The previous reports have indicated the influence of helium pressure and target tissue distance on DNA entrance and tissue damage in plant cells (Rubio et al 2004 ). In nopal, high-pressure systems using 1200 psi and 7.5 cm in callus and apical cultures led to satisfactory transient expression (Llamoca-Zarate et al 1998 , 1999b ). However, higher transient expression levels were found in our investigation when applying a lower helium pressure ( Table 1 ; Fig.…”

“…The osmotic treatment prior to particle bombardment is used to reduce cell damage, enhance transient GUS expression, increase stable transformation, and facilitate cell membrane stabilization after transformation (Suratman et al 2010 ). The previous studies made in nopal utilized a 12–16-h period of exposition prior to particle bombardment, but the osmotic agent was not described (Llamoca-Zarate et al 1999b ). This differs consistently with our findings ( Table 1 ) , since, when the exposition time was increased, we found a statistically significant decrease ( α = 0.05) in foci production.…”

“…This was also observed in plant species like tomato (Camacho-Beltrán 2008 ). Unfortunately, the previous reports in nopal particle bombardment do not describe which plasmid DNA concentration was used (Llamoca-Zarate et al 1998 , 1999b ). On the other hand, limiting the DNA amount used for particle bombardment results in fewer DNA molecules covering an individual particle (Lowe et al 2009 ).…”

“…Since nopal tissue architecture is more complex, this could prevent the penetration of tungsten particles into deeper cell layers, thereby generating a lower transient GUS expression level. Nevertheless, in the previous reports, tungsten particles of 1.3 µm diameter were used during the particle bombardment of nopal apical meristems, generating acceptable results in terms of transient GUS expression (Llamoca-Zarate et al 1999b ). Similar results to the ones reported here were registered in Jatropha curcas , where the use of 900 psi and 0.6 µm gold particles generated a higher survival percentage in bombarded tissues (Joshi et al 2011 ).…”