Transient Gene Expression in Maize, Rice, and Wheat Cells Using an Airgun Apparatus

Abstract

An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNAcoated tungsten particles. The ,-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 x 10-3. In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals.

Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl.) Sw. protoplasts

“…While capacitors with a higher charge accumulation capacity (900 and 1000 µF) gave higher transformation frequencies ( 50%) when used in the range of 2000 to 4000 mJ, capacitors of lower charge accumulation (400 and 800 µF) led to the death of approximately 70% of the protoplasts ( Figure 3A). Maximum transient gene expression has been reported under electric field strengths causing more than 50% reduction in protoplast viability (Fromm et al, 1985;Hauptmann et al, 1987;Oard et al, 1989).…”

“…The transient nature of membrane pores and of structural alterations to the cell wall induced by such electric fields imposes conformational restrictions on the type of macromolecules that can be introduced (Neumann et al, 1982). Nandadeva et al (1999) reported that the physical structure of DNA is critical for efficient transformation, with denatured molecules being much more favorable for transformation, although high transformation efficiency has been obtained with supercoiled plasmid DNA (Bates et al, 1990;Fromm et al, 1985;Oard et al, 1989).…”

Novel Techniques for Gene Delivery into Plants and Its Applications for Disease Resistance in Crops

“…Therefore, a number of modifications have been made to the power source used to propel the microprojectiles to control microprojectile penetration into cells such as regulated nitrogen gas pressure, an air gun and compressed air [24]- [26]. A main difference in the air gun technique is that the DNA is suspended with the microprojectiles rather than being coated on them [25]. The creators of mentioned techniques assumed that the movement of DNA independent of microprojectiles which allows them to target small locations of tissues.…”

Wheat biotechnology: A minireview

“…This method of introducing DNA into cells by physical means was developed to overcome the biological limitations of Agrobacterium and the difficulties associated with plant regeneration from protoplasts. Initial studies on particle bombardment as a gene delivery method achieved transient expression of gus gene following bombardment of cell suspensions (Wang et al 1988), leaf bases and apical tissues (Oard et al 1990), immature embryos (Chibbar et al 1991). Vasil et al (1991) obtained stably transformed callus lines that expressed all the marker genes tested (gus, nptII and EPSPS).…”