The Pilot Study For The Improvement Of Wheatgrass Drought Resistance Using Gene Engineering Techiniques

Wheatgrass is high quality forage. It is a considerable meaning to genetically modify and improve growing properties of wheatgrass. A regeneration system and a bombardment procedure suitable for wheatgrass were developed. In this paper Through these systems, the drought induced transcription factor CBF4 and herbicide-resistance gene Bar were introduced into wheatgrass callus, and drought induced transgenic wheatgrass were obtained. The study in this paper is aimed at establishing and optimizing the regeneration and transformation system using immature spikes and mature embryos of A.mongolicum Keng, A. cristatum cv.'Fairway', A. desertorum cv.'Nordan' and A. cristatum×a.desertorum cv.'hycrest-Mengnong'. The factors that the influence of wheatgrass callus inducing differing and regenerating were primarily discussed. The results were as follows:1. The wheatgrass callus induction and callus differentiation frequency were apparent differences in the same medium with different genotypes, using immature spikes as explants, the feasible genotype is A.cristatum×a.desertorum cv.'hycrest-Mengnong'; however is A.mongolicum Keng the most suitable variety when using mature embryos. 2. The feasible regeneration medium were selected for culturing wheatgrass immature spikes and mature embryos. The feasible medium using immature spikes were: The callus inducing medium: advanced MS+2,4-D(2.0mg/L) Subculture medium: advanced MS+2,4-D(2.0mg/L)+6BA(0.2mg/L) Differentiation medium: MS+KT(3.0mg/L)+NAA(0.5mg/L) Rooting medium: 1/2MS+NAA(0.1mg/L) The feasible medium using mature embryos were: The callus inducing medium: MS+mannite(0.2mol/L)+2,4-D(2.0mg/L) Subculture medium: MS+ mannite (0.2mol/L)+2,4-D(2.0mg/L)+ABA(0.3mg/L) Differentiation medium: MS+KT(3.0mg/L)+NAA(1.0mg/L) Rooting medium: 1/2MS+NAA(0.5mg/L) Four wheatgrass immature spikes and the callus induced by mature embryo were explants using PDS-100He gene gun for transformation. The transformations were confirmed by PCR and RT-PCR. The results showed that immature spikes of bar gene displayed that 18 plantlets , CBF4 displayed that 6 plantlets were positive results. The co-transformation efficiency of both bar and CBF4 was 27.8%. The mature embryos of bar gene displayed that 3 plantlets showed positive results, but no CBF4 gene plantlets showed positive results. The co-transformation efficiency of both bar and CBF4 was 0%.