Protoplast culture and Agrobacterium-mediated transformation of Asparagus officinalis L. using somatic embryogenic cell cultures.

摘要

Plant improvement via biotechnology depends on the ability to regenerate plants from target cells. Protocols based on somatic embryogenesis are ideal for this purpose because somatic embryos originate from single cells and upon germination, a root and shoot are produced from the same structure expediting plant formation. This study examines using embryogenic suspension cultures to expedite plant regeneration from Asparagus officinalis L. protoplasts for potential direct-gene-transfer studies, and determines optimal parameters for transient and stable transformation of asparagus embryogenic suspension cells with Agrobacterium.; The ability of asparagus protoplasts derived from embryogenic suspension cells to undergo direct somatic embryogenesis and germinate into normal plants was influenced by genotype and auxin source. Embryogenic suspensions were initiated from four asparagus genotypes and cultured in either 5 {dollar}mu{dollar}M 2,4-D or 50 {dollar}mu{dollar}M NAA. There was a significant interaction between genotype, suspension auxin, and inclusion or exclusion of PGR's in the protoplast culture media on plating efficiency and somatic embryo formation. Plating efficiencies at 14 days ranged from 0-40% and globular embryos developed from protoplasts in some treatments in the same amount of time. All four genotypes regenerated plants although Rutgers 22 had the highest germination frequency at 42%.; In an effort to increase the Agrobacterium transformation frequency in asparagus, embryogenic suspension cells were targeted and relevant transformation parameters were systematically optimized via transient GUS expression with an intron-containing GUS gene. Embryogenic cells inoculated at 5 {dollar}times{dollar} 10{dollar}sp7{dollar} cfu/ml with either A. tumefaciens strain EHA105 or GV3101 (pMP90) that had been induced with acetosyringone and cocultivated for four days was determine to be optimal for transient GUS expression. Selective agents were compared and the cells were most sensitive to glufosinate followed by G418 and kanamycin, respectively. One transgenic plant was produced when the optimal parameters were applied to stable expression using the BAR gene. Transient GUS expression was low when compared to stable expression. This indicated that the T-DNA was entering the cells and being expressed but not integrated into genomic DNA.