The investigation of optimal bombardment parameters for transient and stable transgene expression in Sorghum

Abstract

This report outlines the development of optimized particle inflow gun (PIG) parameters for producing transgenic sorghum (Sorghum bicolor (L.) Moench). Both transient and stable expression were examined when determining these parameters. The uidA reporter gene (GUS) encoding b-glucuronidase was used in transient experiments and the green fluorescent protein (GFP) used to monitor stable expression. Initially, optimization was conducted using leaf segments, as the generation of sorghum callus in sufficiently large quantities is time-consuming. Following leaf optimization, experiments were conducted using callus, identifying a high similarity between the two tissue types r s 0X83X High levels of GUS expression were observed in both leaf and callus material when most distant from the DNA expulsion point, and using a pressure greater than 1800 kPa. A higher level of expression was also observed when the aperture of the helium inlet valve was constricted. Using the optimized conditions (pressure of 2200 kPa, distance to target tissue of 15 cm from the expulsion point, and the aperture of the helium inlet valve at one full turn), three promoters (Ubiquitin, Actin1 and CaMV 35S) were evaluated over a 72-h period using GUS as the reporter gene. A significantly higher number of GUS foci were counted with the Ubiquitin construct over this period, compared to the Actin1 and CaMV 35S constructs. Stable callus sectors (on 2 mg l 21 bialaphos) with GFP expression were visualized for as long as 6 wk post-bombardment. Using this optimized protocol, several plants were regenerated after having been bombarded with the pAHC20 construct (containing the bar gene), with molecular evidence confirming integration.

Genetic transformation of Sorghum bicolor

“…They include: Agrobacterium-mediated indirect method followed by electroporation, particle bombardment and mild ultrasonication pollen-mediated transformation. Strength of promoters in driving the expression of various genes of interest was also reported (Able et al, 2001;Hill-Ambroz and Weeks, 2001;Jeoung et al, 2002;Tadesse et al, 2003). Emani et al, (2002) indicated that among the cereals, sorghum has been the most recalcitrant crop species to genetic modification and therefore, there are few reports available on sorghum transformation among which the majority have reported transgene silencing.…”

“…Depending upon the plant species being examined, the promoter will also be an important factor in evaluating optimal transgene expression (Able et al, 2001). In general, the CaMV35S promoter is strong in dicot species while its expression level is much lower in monocotyledons.…”

“…During the same year, Able et al, (2001) investigated the transient GUS expression under the control of these promoters (ubiquitin1, actin1 and CaMV35S) and reported that a significantly higher number of GUS spots were obtained with ubiquitin1 promoter when compared to actin1 and CaMV35S promoters. Similar studies were also carried out by Tadesse et al, (2003) in sorghum with immature embryos and shoot tips using four different promoters (ubiquitin1, actin1, adh1 and CaMV35S) for transient GUS expression.…”

“…The second most important reporter system is GFP which acquired importance because of its nondestructive visualization systems that can facilitate the recovery of identified transformed tissues (Gurel et al, 2009;Gao et al, 2005a, b;Jeoung et al, 2004;Able et al, 2001). Green fluorescent protein, comprises of 238 amino acids (26.9 kDa), originally isolated from the jellyfish Aequorea victoria.…”

“…The use of gfp as reporter system in transformation studies along with agronomically important genes proved to be a failure in sorghum as no somatic embryos were formed on the sectors selected on bialophos co-bombarded with gfp and bar genes. Further investigations are needed to ascertain the toxicity of GFP to sorghum cells (Able et al, 2001;Jeoung et al, 2002). The other disadvantage in using GFP is the need for costly equipment like fluorescent stereomicroscope for detection of GFP.…”