Enhancement of leucaena leucocephala tissue regeneration and Agrobacterium-mediated transformation

Abstract

Leucaena leucocephala, (leucaena) is a fast-growing leguminous tree with many positive applications, ranging from biofuel/biomass to animal fodder. This research aims to improve digestibility of leucaena by reducing the concentration of the toxic amino acid mimosine. The approach to accomplish this was transformation of leucaena with isolated genes from Rhizobium sp. strain TAL1145 that are involved in degradation of 3-hydroxy 4 pyridone, which is a precursor of mimosine biosynthesis. The main focus of this work was to improve the existing transformation protocols by overcoming problems with tissue regeneration and DNA transfer, which have been the limiting factors in replicating previous work. Our experimental trials indicated three key limiting factors: (i) production of phenolic exudates by explants, which deter tissue growth, (ii) accumulation of necrotic material at explant cut surface, and (iii) inefficient rooting as a hindrance for regeneration. We hypothesized that overcoming these three specific barriers would improve tissue regeneration and genetic transformation significantly. Reduced production of phenolic exudates was accomplished by introducing 0.8-1.0% activated charcoal to adsorb the phenolics released into the growth media. Introduction of a cell recovery phase and supplementation of the medium with activated charcoal prevented development of necrotic cell material. Proper root induction was achieved through two means: (i) elongation media development: which enabled explants to elongate shoots prior to root induction; and (ii) activated charcoal was utilized as a darkening media agent for improved root induction. Our results thus far have established that prevention of necrotic cell death coupled with timely induction of a healthy root system through darkened media, improves both tissue regeneration and transformation frequency of leucaena. PCR analysis has shown the presence of the transgenes in 16 individuals. These explants originated from four independent Agrobacterium-mediated transformation experiments. This research contributes towards the development of mimosine-free leucaena and also the improvement of transformation protocols for woody legumes.