Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciens-mediated gene transfer to shoot apices

Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selectable marker. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops were thoroughly wetted with PPT solution (2 mg/ml). The multiple axillary shoots developing from the shoot apices were excised onto a medium containing 20 mg/l PPT. The surviving shoots were transferred every second week onto fresh medium containing 20 mg/l PPT. At each transfer, the number of surviving shoots decreased, until it stabilized. Indeed, some of these chimeric shoots surviving the PPT selection, eventually produced new green healthier axillary shoots which could be transferred to soil. This whole process took from 5 to 9 months after co-cultivation. Average transformation frequencies of 2.8% for cv. Unicrop and of 0.4% for the commercial cultivar Merrit were achieved. Molecular analysis of T₀, T₁, and T₂ generations demonstrated stable integration of the foreign gene into the plant genome and expression of the integrated gene. Transformed plants of the T₁ and T₂ generations were resistant in glasshouse trials where the herbicide Basta (0.1 mg/ml) was sprayed onto whole plants. These results demonstrate that Agrobacterium-mediated gene transfer to preorganised meristematic tissue combined with axillary regeneration can form the basis of a routine transformation system for legume crop species which are difficult to regenerate from other explants.     

Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin

Flower color is mainly determined by anthocyanins. Rosa hybrida lacks violet to blue flower varieties due to the absence of delphinidin-based anthocyanins, usually the major constituents of violet and blue flowers, because roses do not possess flavonoid 3',5'-hydoxylase (F3'5'H), a key enzyme for delphinidin biosynthesis. Other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. We analyzed the flavonoid composition of hundreds of rose cultivars and measured the pH of their petal juice in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color change toward blue. Expression of the viola F3'5'H gene in some of the selected cultivars resulted in the accumulation of a high percentage of delphinidin (up to 95%) and a novel bluish flower color. For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, we down-regulated the endogenous dihydroflavonol 4-reductase (DFR) gene and overexpressed the Irisxhollandica DFR gene in addition to the viola F3'5'H gene in a rose cultivar. The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.     

Stability of the Nitrogen Concentration of Inoculated White Lupins during Pod Development

The general pattern of decrease of the 'critical' plant N concentration(i.e. minimum concentration required for maximum growth rate)during growth has been described for several C₃ non grain-legumespecies, and this can be used as a reference curve for diagnosisof N nutrition in these species. The present study was undertakento investigate changes of N concentration during growth of agrain legume, in different conditions of N nutrition. Whitelupin (Lupinus albus L.) was grown for six crop seasons in fieldtrials in which inoculation with Rhizobium lupini, nitrogenfertilizer rate, cultivar and plant density were density weremanipulated. The yield and dry matter production of noninoculatedplants were lower than, or at the best similar to, those ofinoculated plants, whatever the level of N supply. From anthesisto the beginning of seed filling, the N concentration of shootsof inoculated plants was found to be remarkably stable betweenyears, N fertilization regimes, cultivars, and for individualplants within a plot. Nitrogen concentration only varied withplant density. By contrast, the N concentration of noninoculatedplants was highly variable and generally lower than that ofinoculated plants, whatever the level of N supply. The highand stable N concentration of inoculated plants did not appearto be necessary for maximum growth rate but seemed to be requiredfor maximum production of seed dry matter and N. The potentialuse of these results to diagnose, in any white lupin crop, aninefficiency of the lupin-R. lupini interaction is evaluated.Copyright1993, 1999 Academic Press Lupinus albus L., white lupin, N₂ concentration, inoculated plants, non-inoculated plants, N₂ fixation efficiency, diagnosis