Reproducible hairy root transformation and spot-inoculation methods to study root symbioses of pea

Pisum sativum (pea) has been a model organism in plant research for more than a century; however, it has lagged behind other plants in becoming the model organism to study root symbioses, i.e., those resulting in nodulation and mycorrhiza-formation. Reasons for this stem from the fact that pea is a large plant which requires a large area for growth and has a relatively long life-cycle (~ 90 days). It has a large genome (about 4 × 109 base pairs [1]) still to be sequenced, and many molecular techniques, especially those of transformation, are difficult to use [1] to study the function of the genes involved in the nodulation process. Yet, the agricultural and economic importance of pea is significant, especially in Canada, so despite the difficulty in manipulating this species, it is relevant to study its beneficial root symbioses and to capitalize on its large collection of symbiotic mutants [2]. Pea production is expected to increase further as farming practises are focusing on agricultural sustainability, with farmers once again utilizing the benefits of crop rotation to decrease fertilizer applications.Two legumes, Lotus japonicus and Medicago truncatula (barrel medic), have been used as model plants to study the plant-rhizobial and the plant-fungal relationships that lead to the fixation of nitrogen and the increased uptake of phosphorus, respectively [e.g., [3,4]]. These model organisms have helped advance our understanding of the molecular dialogue between plants, rhizobia, and fungi, and the signalling pathway which ensues. The early symbiotic events occurring in the root and leading to nodule organogenesis or to mycorrhiza formation have been a subject of great interest but are difficult to study [e.g., [5,6]]. Complications arise in locating in time and space specific root events, such as those that occur before the microsymbionts have entered the root and those that take place within non-translucent and thick roots after the microsymbiont entry [5,6]. Many