Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Abstract

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.

GENETIC TRANSFORMATION AND REGENERATION OF COMMON BEAN (Phaseolus vulgaris L.) USING Agrobacterium SYSTEM

“…In tobacco, leaves were wounded by microprojectile prior to inoculate with Agrobacterium strain LBA4404 containing nptII gene, and transferred to regeneration medium with 100 mg/l kanamycin for selection (Bidney et al, 1992). High frequency of transformation, based on kanamycin resistance in selection medium containing 100 mg/l kanamycin was obtained from lettuce cotyledon explants co-cultivated with A. tumefaciens vectors (Torres et al, 1993).…”

Transformation of Virus-Resistant Genotype of Gossypium hirsutum L. with Pesticidal Gene.

“…To overcome this barrier of regeneration, particle bombardment method has been used to transform recalcitrant crops (Christou,1992;John and Keller, 1996;McCabe et al, 1988;McCabe and Martinell, 1993). Recently it has been shown that particle bombardment of plant tissues enhances the transformation efficiency through Agrobacterium (Bidney et al, 1992;Finer and McMullen,1990).…”

Transient gene expression in shoot apical meristems of sugarbeet seedlings after particle bombardment

“…To promote the reactivation of cell metabolism, phytohormones, as used by Bidney et al (1992) for enlargement of sunflower menstems, were applied for 24 h prior to bombardment. These hormones are known to enhance the mitotic activity of (Jegla and Sussex, 1989), an increase in mitotic activity of those cells might even promote the recovery of chimeras with transgenic offspring.…”