An efficient and broadly applicable method for transient transformation of plants using vertically aligned carbon nanofiber arrays

Abstract

Transient transformation in plants is a useful process for evaluating gene function. However, there is a scarcity of minimally perturbing methods for gene delivery that can be used on multiple organs, plant species, and non-excised tissues. We pioneered and demonstrated the use of vertically aligned carbon nanofiber (VACNF) arrays to efficiently perform transient transformation of different tissues with DNA constructs in multiple plant species. The VACNFs permeabilize plant tissue transiently to allow molecules into cells without causing a detectable stress response. We successfully delivered DNA into leaves, roots and fruit of five plant species (Arabidopsis, poplar, lettuce, Nicotiana benthamiana, and tomato) and confirmed accumulation of the encoded fluorescent proteins by confocal microscopy. Using this system, it is possible to transiently transform plant cells with both small and large plasmids. The method is successful for species recalcitrant to Agrobacterium-mediated transformation. VACNFs provide simple, reliable means of DNA delivery into a variety of plant organs and species.

Plant Artificial Chromosomes: Construction and Transformation

“…), protection or at least delayed degradation of cargoes by nanoparticles, prolonged active lifespan of cargoes within plant cells, and the potential for transient expression of exogenous genes without integration into the plant genome. , Jean et al demonstrated the effectiveness of vertically aligned carbon nanofiber (VACNF) in transforming a 11 kb Gateway plasmid carrying YFP into Arabidopsis leaves, resulting in detectable fluorescent signals. However, further optimization the characteristics of VACNF are required to achieve the successful transformation of even larger plasmids . As nanomaterial-mediated gene delivery systems continue to evolve, there is hope that they will enable the successful transformation of large DNA fragments into plant cells independently from native chromosomes.…”

“…However, further optimization the characteristics of VACNF are required to achieve the successful transformation of even larger plasmids. 97 As nanomaterial-mediated gene delivery systems continue to evolve, there is hope that they will enable the successful transformation of large DNA fragments into plant cells independently from native chromosomes.…”

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants

“…Previously, Morgan et al 7 demonstrated the use of carbon nanofibers affixed to rigid silicon substrate to transiently transform leaves of lettuce, N. benthamiana, and poplar, and both leaves and roots of Arabidopsis. Although transformations were successful on a variety of organs, fibers were more difficult to apply to plant tissues with curved surfaces, such as roots or fruits.…”

“…To counteract the hydrophobic nature of SU-8, we apply a thin layer of silicon oxide via atomic layer deposition 15 after fibers are embedded in SU-8. Application of fibers on a rigid substrate for biomolecule/dye delivery utilizes the impact force of tweezer tapping described in Davern et al 6 and the on-plant and on-chip methods described in Morgan et al 7 .…”

“…Flexible VACNF films are applied to curved plant surfaces by first semidrying DNA or dye droplets on the film as with the onchip method from Morgan et al 7 and then rolling the films on curved plant surfaces using a small makeup applicator 16 , 17 .…”