Agrobacterium-mediated Transformation Of Suspension Embryogenic Calluses Of Alfalfa And Regeneration Of Plants Containing The NodMDH Gene

Aluminum (Al) is one of the most abundant elements present in the earth's crust. In acidic soils, its toxicity is considered to be the most limiting factor for plant productivity. Based on an estimate of the world's potentially arable land resources, it has been estimated that most of the cultivable area (78.4%)is composed of acid soils. Alfalfa (Medicago Saliva L.) is a most important legume forage crop widely planted all around the world. The development of biotechnology provided a new way to modify the genome of alfalfa and get what man wanted. We established a new transformation system by which use alfalfa's suspension embryogenic callus as a source to get transgenic plants. This system was simple and effective and founded the base of transgenic study of alfalfa. We also transformed the nodMDH gene into the Baoding alfalfa and performed the root elongation experiment. The primary results of our experiment as list below:1. We established the Agrobacterium-mediated transformation system used suspension embryogenic callus for infection. We got high regeneration frequency plant lines from secondary regeneration. We suspension the embryogenic callus in liquid UM at 110~120rpm through which we got large number of dispersed and uniformed embryogenic cells which competent to Agrobacterium. We transformed these embryogenic cells to get transgenic plants thus avoid the hurt to embryogenic ability coming from the infection and selection. We determined the optimal pH and strength of co-cultivated medium and co-cultivation time, found on 1/4 UM, pH 5.3,cocultivated for 3 days reach the best result. We also found 100umol/L AS can promoted the transformation efficiency. We differentiated all kinds of abnormal embryos and secondary embryos and found the embryos coming from the abnormal racicles and hypocotyls were easy to recovered to plantlets.2. We transformed the nodMDH gene into the alfalfa through this system. Transgenic lines anduntransformed controls had similar leaf morphology and flowering patterns. Root elongation in hydroponic solutions showed that the transgenic plant have increased Al tolerance. The transgenic plants have higher relative root elongation under 20umol/L Al stress than untransformed control. After long terms culture under Al stress, the root system of untransformed control was soft and rotted and covered with purple bacterium. We compared root tip, root collar and root-stem transition region and found the transgenic plants have relative normal root system.