A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques.

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

An efficient method for the physical mapping of transgenes in barley using in situ hybridization.

The genetic transformation of crops by particle bombardment and Agrobacterium tumefaciens systems have the potential to complement conventional plant breeding programmes. However, before deployment, transgenic plants need to be characterized in detail, and physical mapping is an integral part of this process. Therefore, it is important to have a highly efficient method for transgene detection by fluorescence in situ hybridization (FISH). This study describes a new approach, which provides efficient control of probe length and labelling, both of which play an important role in in situ hybridization of transgenes. The approach is based on reducing the size of the plasmid prior to labelling by nick translation, rather than using the whole or linearized plasmid, or varying the amounts of DNaseI in the nick translation mixture. This provided much more efficient labelling of the probe, which yielded optimal hybridization. minimal fluorescent background, and accurate physical location of the transgene.

Protoplast microinjection using agarose microdrops.

A number of plant species have now been transformed, but there remains considerable uncertainty regarding the optimal procedure to be adopted. Agmbacterium species have been used as vectors for the DNA in some instances, whereas in others direct uptake has been achieved following chemical, physical, or electrical treatment of protoplasts or even of cells (see Chapters 27 - 30 of this volume). Viruses are another vehicle being considered in this context. One of the most effective techniques of transforming animal cells is that of microinjection, and this, together with the recent claim of very high rates of transformation of plant cells by microinjection (1), has heightened interest in the more general exploitation of this method of introducing DNA to plants. The technique is now sufficiently well developed to enable it to be more generally adopted, and this chapter will describe the method that we have developed. An important feature of the method is the use of low melting point (LMP) agarose, both for holding protoplasts during microinjection and for their subsequent culture. The method will be described in detail for tobacco mesophyll protoplasts, and then variations of the method for rape hypocotyl and sugar beet suspension culture protoplasts will be listed.