Transformation of recalcitrant species

Cereals, legumes, and woody plants are commonly categorized as recalcitrant to transformation. However, the hypothesis that some plants lack the biological capacity to respond to essential triggers for integrative transformation, or have cellular mechanisms preventing integrative transformation, can effectively be rejected. Broadly applicable selection methods are well established and the key to transform recalcitrant species appears to be the development of methods to expose many regenerable cells to nondestructive gene transfer treatments.

Knowledge of the relative susceptibility of different cells and tissues to transformation by Agrobacterium tumefaciens, would be helpful in devising strategies for transformation experiments for recalcitrant plant species. Although we know much about the contribution of the bacterium, we know little about its interaction with the plant cell and about the events surrounding gene transfer. It is known that Agrobacterium DNA transfer is highly regulated and is triggered only in the presence of susceptible cells of the plant host. However, does Agrobacterium select between cell types? What features determine favored cells for gene transfer? Are there physiological requirements for efficient T-DNA integration? Can wound response of recalcitrant plant species efficiently induce the expression of vir genes existing in the Ti plasmid of Agrobacterium?

A clear understanding of the factors determining the amenability of the transformed cells for regeneration will also favorably contribute to overcome the problem of transforming recalcitrant species. Despite a vast lore of information on hormonal control, largely arrived at through trial and error, knowledge of the fundamental biology underlying induction of plant regeneration and organogenesis remains scanty. For example, gene expression associated with organ-specific inductive events is poorly characterized and the mechanism(s) by which growth factors such as auxins and cytokinins act to induce organogenesis is still a mystery. In a developmental perspective, it has been suggested that plant tissues are composed of cell populations with different states of developmental competence. [86] Although this implies that cells belonging to different populations have different fates, the major issue remains as to the molecular characterization of the different developmental states of the cell and the determination of organogenic ‘markers’. Additionally, what makes a cell competent for dedifferentiation, proliferation and regeneration?

Protocols aimed to avoid long tissue culture- and hormone-dependent regeneration processes have been developed which are based on the natural capability of plants for spontaneous regeneration. These protocols, which are characterized by the requirement of a limited number of plant manipulations, proved to be successful for the stable transformation of plants acting as important model systems in fundamental research (ie. Arabidopsis thaliana, [87] and for the transformation of crops such as tomato. [88] These protocols should be applicable for the genetic engineering of recalcitrant plant species such as bell pepper where transformation, [89–91]) has been limited because of the difficulties of developing an efficient and universal plant regeneration system. The regeneration of bell pepper has been performed using empirically determined combinations of growth regulators. [92–6] However, protocols for spontaneous plant regeneration have been applied to different cultivars of bell pepper which proved to be efficient. [97–9] Some of these protocols, combined with Agrobacterium tumefaciens mediated gene transfer and selection, have been shown to be effective in regenerating stable transformed plants of tomato and they are also promising tools to transform bell pepper.