Genetic transformation of Leucaena leucocephala for reduction of mimosine content

Leucaena leucocephala (leucaena) is an important leguminous tree for agroforestry in many tropical and subtropical countries. Leucaena can grow under different soil conditions, such as alkaline and arid regions, and it is highly resistant to insects and diseases. Leucaena is known as 'the alfalfa of the tropics' due to the high protein content of its foliage. However, leucaena plants contain a toxin, mimosine, which is harmful if consumed by animals. Recently, we have isolated the genes required for mimosine catabolism from the mimosine-degrading Rhizobium strain TAL1145 that nodulates leucaena. We have identified pydA and pydB as the two structural genes required for degradation of 3-hydroxy-4-pyridone (HP), which is a precursor in the biosynthesis of mimosine and also an intermediate product of mimosine degradation. A dioxygenase, encoded by pydA, is involved in a meta-cleavage reaction, opening the aromatic ring of HP while a hydrolase, encoded by pydB, is involved in further hydrolysis of the degradation product. There are two main goals for this project; first we aim to develop an Agrobacterium-mediated transformation and regeneration protocol for leucaena, and second, we intend to express the Rhizobium genes for HP degradation in the transgenic leucaena plants. We hypothesize that transgenic leucaena plants expressing these genes will be low in mimosine content, and therefore more suitable as an animal fodder. As most woody plants, leucaena is highly recalcitrant to genetic engineering; however, we were successful at developing an Agrobacterium-mediated transformation method for leucaena, achieving a transformation efficiency of 2%. After achieving a reliable transformation protocol for leucaena, we constructed four different binary plasmids containing the Rhizobium genes pydA, pydB, and pydA/pydB fusion and used them to generate 15 transgenic leucaena plants expressing the bacterial dioxygenase and hydrolase. Through high performance liquid chromatography (HPLC) analysis it was determined that mimosine content was decreased by approximately 46% in the transgenic plants expressing both pydA and pydB genes together, while there was little or no decrease in mimosine content in the transgenic plants carrying either pydA or pydB genes alone.