An approach to overcoming regeneration recalcitrance in genetic transformation of lupins and other legumes

For pulse legume research to fully capitalise on developments in plant molecular genetics, a high throughput genetic transformation methodology is required. In Western Australia the dominant grain legume is Lupinus angustifolius L. (narrow leafed lupin; NLL). Standard transformation methodology utilising Agrobacterium tumefaciens on wounded NLL seedling shoot apices, in combination with two different herbicide selections (phosphinothricin and glyphosate) is time consuming, inefficient, and produces chimeric shoots that often fail to yield transgenic progeny. Investigation of hygromycin as an alternative selection in combination with expression of green fluorescent protein indicated that transformation of NLL apical cells was not the rate limiting step to achieve transgenic shoot materials. In this research it was identified that despite ready transformation, apical cells were not competent to regenerate. However a deep and broad wounding procedure to expose underlying axillary shoot and vascular cells to Agrobacterium, in combination with delayed selection proved successful, increasing initial explants transformation efficiency up to 75?% and generating axillary shoots with significant transgenic content. Based on knowledge gained from studies of plant chimeras, further subculture of these initial axillary shoots will result in development of low chimeric transgenic materials with heritable content. Furthermore, the method was also tested successfully on other Lupinus species, faba bea and field pea. These results demonstrate that development of a high yielding transformation methodology for pulse legume crops is achievable.     

Phospholipase Dε enhances Braasca napus growth and seed production in response to nitrogen availability

Phospholipase D (PLD), which hydrolyses phospholipids to produce phosphatidic acid, has been implicated in plant response to macronutrient availability in Arabidopsis. This study investigated the effect of increased PLDε expression on nitrogen utilization in Brassica napus to explore the application of PLDε manipulation to crop improvement. In addition, changes in membrane lipid species in response to nitrogen availability were determined in the oil seed crop. Multiple PLDε over expression (PLDε‐OE) lines displayed enhanced biomass accumulation under nitrogen‐deficient and nitrogen‐replete conditions. PLDε‐OE plants in the field produced more seeds than wild‐type plants but have no impact on seed oil content. Compared with wild‐type plants, PLDε‐OE plants were enhanced in nitrate transporter expression, uptake and reduction, whereas the activity of nitrite reductase was higher under nitrogen‐depleted, but not at nitrogen‐replete conditions. The level of nitrogen altered membrane glycerolipid metabolism, with greater impacts on young than mature leaves. The data indicate increased expression of PLDε has the potential to improve crop plant growth and production under nitrogen‐depleted and nitrogen‐replete conditions.