Biolistic transformation of cotton zygotic embryo meristem

Biolistic transformation of cotton (Gossypium hirsutum L.) meristems, isolated from mature seed, is detailed in this report. A commercially available, helium-driven biolistic device (Bio-Rad PDS1000/He) was used to bombard gold particles coated with a marker gene (uidA or "β-glucuronidase") into the shoot meristem. The penetration of gold particles was dependent on bombardment parameters and it was mostly one to two cell layers deep. Stable transformation of epidermal L1 layer was consistently observed in approximately 5% of the seedlings. Germ line transformation was observed in up to 0.71% of bombarded meristems by several laboratories. Using this method identification of germ line transformation is laborious and time-consuming. However, the protocol described here represents a simple and efficient method for generating germ line transformation events. In addition, this procedure offers a quick method to evaluate gene constructs in cotton tissues (embryos, cotyledons, leaf), especially fibers which originate as single cells from the maternal epidermis layer.