Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures.

Abstract

Genetic transformation of cereals by direct DNA delivery via microprojectile bombardment has become an established procedure in recent years. But the derivation of functional transgenic plants, especially in wheat, is still problematic, mainly due to low efficiency of DNA delivery and the reduced regeneration capability of microprojectile-bombarded tissue. We focussed on these two aspects and found that the regeneration of scutellar calli of wheat can be rendered highly efficient and considerably accelerated by a liquid culture phase in screen rafts. We also found that the expression of a reporter gene following DNA delivery by microprojectile can be improved by maintaining the scutellar calli in 0.25 M mannitol before and after bombardment, by bombardment in the presence of silver thiosulfate and Ca(NO3)2 (rather than CaCl2) and by the elimination of spermidine from the DNA/microprojectile mixture. A protocol that includes all these features leads to several-fold higher transient expression of the reporter gene than have previously published procedures.