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Trends in Biotechnology 




Nanoparticle-Mediated Delivery towards 
Advancing Plant Genetic Engineering 

Francis J. Cunningham , 1 ' 6 Natalie S. Goh , 1,6 Gozde S. Demirer , 1 Juliana L. Matos , 2,3 and 
Markita P. Landry 1,3A5 -*' @ 

Genetic engineering of plants has enhanced crop productivity in the face of 
climate change and a growing global population by conferring desirable genetic 
traits to agricultural crops. Efficient genetic transformation in plants remains a 
challenge due to the cell wall, a barrier to exogenous biomolecule delivery. 
Conventional delivery methods are inefficient, damaging to tissue, or are only 
effective in a limited number of plant species. Nanoparticles are promising 
materials for biomolecule delivery, owing to their ability to traverse plant cell 
walls without external force and highly tunable physicochemical properties for 
diverse cargo conjugation and broad host range applicability. With the advent of 
engineered nuclease biotechnologies, we discuss the potential of nanoparticles 
as an optimal platform to deliver biomolecules to plants for genetic engineering. 

Current Biomolecule Delivery Methods for Genetic Engineering in Plants 

Food security has been threatened with decreasing crop yields and increasing food consumption 
in the wake of population growth, climate change, increasing shortage of arable land, and crop 
usage as raw materials [1,2], Classical plant breeding to obtain plants with preferred genotypes 
requires crossing and selection of multiple plant generations, which disallows introduction of traits 
that do not currently exist in the species. A technique that enables specific horizontal gene transfer 
stands to greatly benefit the agricultural industry by conferring desirable traits to plants, such as 
increased yield, abiotic stress tolerance, and disease and pest resistance [3]. 

Genetic engineering has recently seen major advances in animal systems, though progress has 
lagged in plants. When compared to the numerous and diverse gene and protein delivery 
methods developed for animal systems, significantly fewer methods exist for plants (Figure 1 , 
Key Figure). Broadly, modern genetic transformation of plants entails two major steps: genetic 
cargo delivery and regeneration of the transformed plant, the necessity and difficulty of the latter 
being highly dependent on what delivery method is used and whether stable transformation is 
desired. Regeneration procedures involve three parts: the induction of competent totipotent 
tissue, tissue culture to form calli (see Glossary), and selection and progeny segregation. 
Regeneration protocols are dominated by complex hormone mixtures, which are heavily 
species and tissue dependent, making protocol optimization the key to increasing procedure 
efficacy. The challenge of genetic cargo delivery to plants is attributed to the presence of the 
multilayered and rigid plant cell wall, otherwise absent in animal cells, which poses an additional 
physical barrier for intracellular delivery of biomolecules and is one of the key reasons for the 
slower implementation and employment of genetic engineering tools in plants [4j. 

Amongst conventional plant biomolecule delivery approaches, Agrobacterium -mediated and 
biolistic particle delivery are the two most established and preferred tools for plant genetic 
transformations (Box 1). Current biomolecule delivery methods to plants experience challenges 


Plant biotechnology is key to ensuring 
food and energy security; however, 
biomolecule delivery and progeny 
regeneration continue to be key chal¬ 
lenges in plant genetic engineering. 

Conventional biomolecule delivery 
methods in plants have critical draw¬ 
backs, such as low efficiency, narrow 
species range, limited cargo types, 
and tissue damage. 

Advances in nanotechnology have cre¬ 
ated opportunities to overcome limita¬ 
tions in conventional methods: 
nanoparticles are promising for spe¬ 
cies-independent passive delivery of 
DNA, RNA, and proteins. 

The advent of nuclease-based gen¬ 
ome editing (eg., CRISPR-Cas9) has 
ushered in a new era of precise genetic 
engineering that, among other 
impacts, has enabled the development 
of genetically engineered crops with¬ 
out harsh regulatory restrictions. 

The potential of nanoparticles to over¬ 
come limitations in conventional deliv¬ 
ery makes them excellent candidates 
for delivery of nuclease-based genome 
editing cargo, thus making nanoparti¬ 
cle delivery a critical technology for the 
advancement of plant genetic 

department of Chemical and 
Biomolecular Engineering, University 
of California Berkeley, Berkeley, CA 
94720, USA 

department of Plant and Microbial 
Biology, University of California, 
Berkeley, CA 94720, USA 
innovative Genomics Institute (IGI), 
Berkeley, CA 94720, USA 

882 Trends in Biotechnology, September 20f 8, Vol. 36, No. 9 https://doi.Org/10.10f6/j.tibtech.2018.03.009 

© 2018 Elsevier Ltd. All rights reserved. 

Trends in Biotechnology 

that hinder their scope of use (Table 1). Methods such as electroporation, biolistics, Agro¬ 
bacterium -mediated delivery, or cationic delivery typically target immature plant tissue (calli, 
meristems, or embryos). These methods require the regeneration of genetically modified 
progeny plants, which can be time-consuming and challenging, whereby efficient protocols 
have only been developed for a narrow range of plant species. Biolistic particle delivery 
circumvents the cell wall via mechanical force, but often damages portions of target tissue 
in the process and yields low levels of gene expression that is often sparse and sporadic. 
Agrobacterium- mediated delivery is subject to orthogonal challenges, the largest being that 
Agrobacterium displays narrow host and tissue specificity, even between specific cultivars of 
the same species [5], Agrobacterium generally experiences lower transformation efficiency for 
both delivery and regeneration in monocotyledonous plants (monocots) over dicotyledon¬ 
ous plants (dicots). Additionally, Agrobacterium yields random DNA integration, which can 
cause disruption of important genes, or insertion into sections of the genome with poor or 
unstable expression [6]. Random DNA integration, however, can be prevented by utilizing 
magnifection with nonintegrating viruses [7], or by using a plasmid deficient in transfer DNA(T- 
DNA) insertion [8]. 

In sum, plant genetic engineering has lagged behind progress in animal systems; conventional 
methods of biomolecule delivery to plants remain challenged by intracellular transport through 
cell walls, and in turn limit plant genetic transformation efficacy. To date, plant biotechnology 
lacks a method that allows passive delivery of diverse biomolecules into a broad range of 
plant phenotypes and species without the aid of external force and without causing tissue 
damage. We posit nanotechnology as a key driver in the creation of a transformational tool to 
address delivery challenges and enhance the utility of plant genetic engineering. 

Nanoparticle-Mediated Biomolecule Delivery in Animal Systems 

Nanoparticles as Molecular Transporters in Living Systems 

Nanotechnology has advanced a variety of fields, including manufacturing, energy, and 
medicine. Of particular interest is the use of nanoparticles (NPs) (Box 2) as molecular trans¬ 
porters in cells, an area that has largely focused on molecular delivery in animal systems. NPs 
allow manipulation on a subcellular level, giving rise to a previously unattainable degree of 
control over exogenous interactions with biological systems. Therefore, the impact of NPs as 
drug and gene delivery vehicles in animals has been nothing short of revolutionary. 

The small size of NPs and their highly tunable chemical and physical properties have enabled 
NP engineering for NPs to bypass biological barriers and even localize NPs in subcellular 
domains of CHO and HeLa cells, among others [10-13]. NPs serve as nonviral, biocompatible, 
and noncytotoxic vectors that can transport a range of biomolecules [small molecules, DNA, 
siRNA, miRNA, proteins, and ribonucleoproteins (RNPs)] [14-19] to biological cells. To this end, 
various features of NPs, including size, shape, functionalization, tensile strength, aspect ratio, 
and charge, have been tuned for efficient intracellular biomolecule delivery to animal systems. 
Furthermore, ‘smart’ NPs have been developed to achieve responsive release of cargo for 
increased control of site-specificity [20]. Various NPs have been manufactured and are 
responsive to a range of stimuli, including temperature [21], pH [22], redox [23], and the 
presence of enzymes [24], 

Outlook and Implications for Nanocarriers in Plant Science 

In contrast to the proliferate studies demonstrating NP-mediated delivery in animals, analogous 
research in plants is relatively sparse (Figure 1 ), owing to the transport challenge imposed by the plant 
cell wall, which renders biomolecule delivery more challenging than for most mammalian systems. 



California Institute for Quantitative 
Biosciences (QB3), University of 
California Berkeley, Berkeley, CA 
94720, USA 

5 Chan-Zuckerberg Biohub, San 
Francisco, CA 94158, USA 
6 These authors contributed equally to 
this work 

@ Twitter: @l_andry_lab 

Correspondence: (M.P. Landry). 

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Nevertheless, knowledge gained from biomolecule delivery to animals provides a blueprint for 
translation to plant systems, and could accelerate advancements in NP-mediated plant biomolecule 
delivery. NP-mediated delivery may overcome the three foremost limitations of current delivery 
techniques in plant systems by controlling NP size to traverse the cell wall, tuning charge and surface 
properties to carry diverse cargo, and greater breadth in utility across plant species. 

NP-mediated delivery in animals has successfully carried many types of cargo indiscriminately, 
whereby certain methods for plants, such as Agrobacterium, can only deliver DNA. For instance, 
Wang and colleagues report NP-mediated RNP delivery to mammalian cells via lipid encapsulation 
[25]. Additionally, plastid engineering is not achievable with Agrobacterium, which only targets the 
plant nuclear genome and cannot target the chloroplast or mitochondrial genomes. Conversely, 
targeting moieties can be attached to NPs to obtain subcellular localization and modification of the 
desired genome. Hoshino and coworkers demonstrate the delivery of quantum dots to the 
nucleus and mitochondria of Vero kidney cells using respective localizing signal peptides [26]. 
Active targeting and controlled release is not achievable with conventional plant biomolecule 
delivery methods, but has been demonstrated in animal systems with NP-based delivery. Davis 
and colleagues designed a polymeric NP with a human transferrin protein-targeting ligand and 
polyethylene-glycol (PEG) on the NP exterior to deliver siRNA to human melanoma tumor cells, 
specifically [15]. Additionally, Lai and coworkers accomplished stimuli-responsive controlled 
release of drug molecules and neurotransmitters encapsulated within mesoporous silica NPs 
(MSNs) to neuroglial cells [27]. Drawing inspiration from progress in NP-mediated delivery for 
animal systems, NP-mediated controlled delivery and release of biomolecules without species 
limitations in plants is a forthcoming goal. 

NP-Mediated Biomolecule Delivery to Plants 

NP-Plant Interactions 

To date, most literature on NP-plant systems focuses on plant-based metallic nanomaterial 
synthesis [28], agrochemical delivery [29], and NP uptake, showing both valuable and deleterious 
effects on plant growth [30,31 ]. Dicot and monocot plants exhibit variable degrees of direct uptake 
of many NP types, including MSNs [32], carbon nanotubes (CNTs) [33], quantum dots [34], and 
metal/metal oxide NPs [35-37]. Once uptaken, certain types of NPs exhibit phytotoxicity via 
vascular blockage, oxidative stress, or DNA structural damage [30]. Conversely, NPs have been 
shown to improve root and leaf growth, and chloroplast production [31]. Tradeoffs between 
phytotoxicity and growth enhancement as a function of species, growth conditions, NP proper¬ 
ties, and dosage are not well understood and call for more studies with afocus on NP physical and 
chemical properties. Closing the knowledge gap in plant physiological response to NP uptake is 
important and should be pursued in parallel with the enhancement of plant science using 
engineered nanomaterials, as the ‘nanorevolution’ in targeted delivery to animals suggests 
tremendous potential for analogous progress in plants. 

Heuristics for Nanocarrier Design 

While a complete structure-function landscape of physical and chemical NP properties that drive 
cargo loading and cellular internalization remains elusive, a heuristic approach to nanocarrier design 
is a useful starting point. NP uptake and transport throughout plant tissue is limited by pore 
diameters, setting size exclusion limits (SELs) for various tissues and organs that are discussed 
extensively in the literature [30,38-43]. The cell wall is commonly thought to exclude particles >5- 
20 nm, although recently NPs up to 50 nm in diameter have been reported as cell wall-permeable 
through unclear mechanisms [38,41]. For genetic engineering applications, where cytosolic or 
nuclear localization is necessary to affect gene function, the plasma and nuclear membranes pose 
additional barriers to delivery. In practice, the cell wall (SEL <50 nm) plays a dominant role in NP size 


Callus: a mass of undifferentiated 
cells that can be used to regenerate 

Cultlvars: short for cultivated 
varieties, a group of plants with 
desired characteristics that have 
been selected from a naturally 
occurring species and are passed 
through propagation. 
Dicotyledonous plants: one of the 
two major groups of flowering plants. 
The eponymous term originates from 
the presence of two embryonic 
leaves upon germination. 

Additionally, dicots can be 
distinguished from monocots by a 
number of characteristics that 
include leaf veins, vascular bundles, 
root development, floral bundles, and 
pollen. See monocotyledonous 

Electroporation: a physical 
transfection method where an 
electric field is applied to create 
temporary pores in cell membranes 
for the uptake of genetic cargo into a 

Explant: any segment of a plant that 
is removed to initiate a culture. 

In planta: a transformation paradigm 
involving the genetic transformation 
of any segment of a plant without 
the need for tissue culture and 

Magnifection: delivering virus 
vectors using Agrobacterium T-DNA 

Meristems: regions of tissue 
containing undifferentiated cells. 

Monocotyledonous plants: one of 

the two major groups of flowering 
plants that have one embryonic leaf 
upon germination. Monocots include 
crops that make up the majority of a 
balanced diet, such as rice, wheat, 
and barley. See dicotyledonous 

Passive delivery: transport of cargo 
across cell wall and membrane to an 
intracellular location without the use 
of mechanical force. 

Protoplasts: plant cells with their 
cell walls removed, typically through 
either mechanical or enzymatic 

Recalcitrant: a species of plant that 
is difficult to genetically transform 
and regenerate into mature plants. 
Often used in the context of 

884 Trends in Biotechnology. September 2018, Vol. 36, No. 9 

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internalization limitations, as the cell membrane SEL is much larger (>500 nm) [38]. NP charge and 
shape greatly influence cell membrane translocation and thus these properties are central to 
nanocarrier optimization [44], Plant cellular uptake can occur through energy-dependent (endocy- 
tosis) and energy-independent (direct penetration) pathways that are not well understood. It is 
commonly reported that internalization is faster and more efficient for cationic NPs versus anionic 
NPs, due to cationic NP binding with the negatively charged cell membrane [44], This charge 
preference has been demonstrated in protoplasts and walled plant cells [45,46]. 

Endosomal escape is critical for subcellular delivery, as vesicle-entrapped NPs can be trafficked for 
degradation or exocytosis, and remain inaccessible for downstream processing if trapped in the 
endosome. Subcellular localization of NPs in plants is not well understood but will depend on the 
uptake pathway, as endocytic proteins and vesicle cargo play a role in endosome fate [47], whereby 
direct cell penetration bypasses endosomal vesicle formation entirely. Serag and colleagues report 
CNT internalization in protoplasts through both direct penetration and endocytosis, supporting prior 
demonstrations in mammalian cells that high aspect ratio NPs undergo vesicle-free internalization 
[48,49]. However, for Serag and colleagues, direct penetration was only observed for cell wall- 
impermeable multiwalled CNTs in protoplasts [48,49], motivating further studies for plant cell wall 
internalization by high aspect ratio NPs. Wong and colleagues have demonstrated passive internali¬ 
zation of single-walled CNTs in extracted chloroplasts [1 29] through a mechanism dependent on NP 
size and zeta potential [130]. Cationic, pH-buffering polymers are well-known endosome disruption 
agents [50] that can function as ligands to improve endosomal escape. Chang and colleagues report 
energy-independent internalization to walled root cells by organically functionalized spherical MSNs 
[51 ]. Notably, endocytosed single-walled CNTs in plants are trafficked to vacuoles but localize in the 
cytosol when loaded with DNA [33,48]. 

Most NPs are amenable to surface adsorption (physisorption) of biomolecules as a simple conju¬ 
gation strategy. However, physisorption may be unstable depending on the specific NP and cargo, 
and thus electrostatic interactions are preferablefor noncovalent cargo loading [52], Cationic surface 
chemistry not only enhances endocytic uptake and escape, but is also amenable to electrostatic 
loading of genetic cargo via attraction with negatively charged DNA and RNA. Covalent NP surface 
functionalization is typically achieved by one of many of ‘click’ chemistries [53]. Notably, covalent 
attachment of thiolated DNA and proteins to gold NPs has shown recent success [54] but the field 
remains open to new strategies for covalent bioconjugation, especially for applications in plants. As 
an alternative to surface functionalization, porous NPs such as MSNs can be internally loaded with 
macromolecules or small chemicals alike, for controlled intracellular release [55]. 

NPs with some or all of the properties mentioned above have demonstrated successful biomolecule 
delivery in plants and are good starting points for choosing the appropriate NP, ligand, and cargo for a 
given application. However, it should be noted that nanocarrier design is a complex, multivariable 
optimization process, such that success will likely require tweaking of these heuristics for different 
systems until a complete NP structure-function relationship is established for plant systems. 

Nanomaterials for Plant Genetic Engineering 

NPs are valuable materials for intracellular biomolecule delivery, owing to their ability to cross 
biological membranes, protect and release diverse cargoes, and achieve multifaceted targeting 
via chemical and physical tunability. Such properties have enabled NPs to revolutionize targeted 
delivery and controlled release in mammalian systems. However, nanocarrier delivery in plants 
remains largely underexplored due to the cell wall, which is typically overcome by chemical or 
mechanical aid (Figure 1). Passive biomolecule delivery to plants is promising for minimally 
invasive, species-independent, in vivo genetic engineering of plants, especially for transient 



Transgene: a gene taken from an 
organism and transferred into the 
genome of another. Consequently, 
transgene integration results in 
transgenic plants. 

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Key Figure 

Nanoparticle (NP)-Mediated Genetic Cargo Delivery to Animals and Plants 

(a) NPs classes commonly employed in genetic cargo delivery 


Bio-inspired NPs 

• Calcium phosphate 

• Chitosan 

• Liposomes 

Genetic cargo delivered a-d 




DNA RNA Protein RNP 

Carbon-based NPs 

• Single-walled carbon 
Multiwalled carbon nanotubes 
• Fullerenes 

Genetic cargo delivered e g 

DNA RNA Protein RNP 

Genetic cargo has been delivered in: 
^ Both animal and plant systems 
O Animal systems only 

£ Plant systems only 

ftl—T \ V' 

yj Neither system 

Silicon-based NPs 

• Silica spheres 
Mesoporous silica NPs 


• Silicon carbide 
Genetic cargo delivered h-k 






(b) Modes of NP-mediated cargo delivery 

Metallic / Magnetic NPs 

• Gold 
■ Silver 
Iron oxide 


Genetic cargo delivered 1 

DNA RNA Protein RNP 

Polymeric NPs 

> Polyethylene-glycol (PEG) 

• Poly(lactic-co-glycolic acid) 

• Polyethylenimine (PEI) 

Genetic cargo delivered l-s 



Protein RNP 

Trends in Biotechnology 

Figure 1. (A) NPs commonly used for biomolecule delivery in both animal and plant systems cover five major categories: bio-inspired, carbon-based, silicon-based, 
polymeric, and metallic/magnetic. We provide a visual comparison of delivery of various genetic cargo [DNA, RNA, proteins (site-specific recombinases or nucleases), 
and ribonucleoprotein (RNP)] with each of the five NP types across animal and plant systems. It is evident that NP-mediated delivery has been utilized with a greater 
variety of genetic cargo in animals than in plants, (b) NP-mediated cargo delivery is conducted via several means. Physical methods include creating transient pores in 
the cell membrane with electric fields, soundwaves, or light, magnetofection, microinjection, and biolistio particle delivery. Nonphysical methods include the use of 

cationic carriers, incubation, and infiltration. a [64], b [86], c [87], d [88], e [89], f [68], g [90], h [91], '[45], '[92], k [58], '[93], m [94], n [95], °[96], p [97], q [98], r [99], s [81], ’[1 00], u [63], 
v [101], W [1 02], x [54]. 

886 Trends in Biotechnology, September 2018, Vol. 36, No. 9 

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Box 1. Common Gene Delivery Methods in Plants 

Agrobacterium-Mediated Transformation 

Agrobacterium tumefaciens is a soil bacterium that infects a wide range of dicots, causing crown gall disease. The 
formation of a gall on the host plant is achieved via the stable transfer, integration, and expression of bacterial DNA in 
infected plants. Engineering of the Agrobacterium plasmid by substitution of the gall-inducing virulence genes with 
genes of interest confers the ability of Agrobacterium to transform the host plant. For this reason, Agrobacterium has 
been harnessed as a tool for plant genetic transformation since the early 1980s [107], 

Genetic transformation occurs through a process involving T-DNA export, targeting, and insertion into the plant nuclear 
genome. The export of T-DNA from the bacterium to the plant cell is facilitated by the activity of virulence genes present 
in the tumor inducing-plasmid of Agrobacterium, but are not themselves transferred. These virulence genes are 
expressed in the presence of phenolic inducers, such as acetosyringone, produced by wounded plant cells. Agro¬ 
bacterium attaches to plant cells, where border sequences on either side of the T-strand (a single-stranded copy of the 
T-DNA sequence) are cleaved. The T-strand is then carried by a transporter with a nuclear localization sequence and 
integrated into the plant nuclear genome. Integration occurs at random positions in the genome via nonhomologous 
recombination, a repair pathway for double-stranded breaks in DNA. 

Gene Gun-Mediated Transformation 

A form of biolistic particle delivery (also called particle bombardment), the gene gun, is a physical method that is 
commonly utilized for plant genetic transformations. Developed in 1982 by Sanford and colleagues [1 08], the process 
involves gold or tungsten microparticles (or microcarriers) coated with genetic cargo that are accelerated by pressurized 
helium (He) gas into plant cells, rupturing cell walls and membranes. The gene gun consists of three main parts: a rupture 
disk, macrocarrier (holding microcarrier particles), and stopping screen. The rupture disk is a membrane designed to 
burst at a critical pressure of He gas. When He gas is accelerated to the desired pressure, the rupture disk bursts, 
creating a shock wave that propels the macrocarrier towards the plant cells. The macrocarrier’s momentum is stopped 
by the stopping screen, which allows genetic cargo-loaded microcarriers to pass and enter the plant cells. 

Unlike Agrobacterium-mediated transformation, biolistic delivery can result in transformation of the nuclear, plastidal, or 
mitochondrial genomes due to the nonspecific localization of genetic cargo. Consequently, more DNA needs to be 
delivered with biolistic delivery than Agrobacterium-mediatecl delivery when targeting the nuclear genome. 

expression in somatic tissue (Table 2). The potential of NP-based plant delivery methods is 
underscored by the limitations of in vitro plant studies in general, wherein regeneration capacity 
varies widely across species, genotype, and even within a single plant depending on develop¬ 
mental age of source tissue [56]. Currently, stable transformation requires progeny regeneration 
from embryogenic calli regardless of the delivery method (Table 2). Thus, parallel optimization of 
delivery and regeneration is necessary to improve efficiency and expand stable transformation 
capabilities to all plant species. 

In 2007, Torney and colleagues were the first to demonstrate NP co-delivery of DNA and chemicals 
to Nicotians tabacunn plants via biolistic delivery of 100-200-nm gold-capped MSNs [45]. In this 
study, a chemical expression inducer was loaded into MSN pores (~3 nm) that were subsequently 
covalently capped with gold NPs. The capped MSNs were then coated with GFP plasmids and 
delivered by gene gun to N. tabacum cotyledons, wherein GFP expression was triggered upon 
uncapping and release of the expression inducer [45]. This seminal paper demonstrated proof of 
concept that strategies common for NP delivery of DNA to mammalian systems can be adapted to 
plants. Notably, gold MSNs were also used for biolistic co-delivery of DNA and proteins, namely 
GFP and Cre-recombinase, demonstrating the ability of MSNs to deliver proteins for gene editing 
[58]. Many delivery strategies still require a gene gun, electromagnetic field, or protoplast PEG- 
transfection [58-63] as NP structure-function parameters have not yet been fully optimized to 
passively bypass the cell wall (T able 3). However, for systems where mechanical or chemical aid is 
necessary for NP internalization, the small size and high surface area of nanocarriers still offers 

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888 Trends in Biotechnology, September 2018, Vol. 36, No. 9 

Table 1. Scope of Use Summary for Plant Biomolecule Delivery Methods 

Delivery method 

Adverse effects of delivery 

Target species/tissue 

Cargo type and size 3 



Biolistic particle- 
(gene gun) 
mediated delivery 

Damage to target tissue & cargo, low 
penetration depth, random 

Depends on tissue type b / 
oalli, embryos, leaves 

DNA, siRNA, miRNA, 
(RNPs), large cargo 

Targeting leaves requires detachment from 
plant, which limits time to observe delivery 
effects; targeting embryos requires laborious 
regeneration protocols, the effectiveness of 
which is highly species/cultivar-dependent 


Damage to target tissue, nonspecific 
transport of material through pores 
may lead to improper cell function 

Unlimited/protoplasts 0 , 
meristems, pollen grains 

Nucleic acids (DNA, 

SiRNA, miRNA) 

Limited cargo-carrying capacity 




High charge densities induce 

Species amenable to 
protoplast regeneration/ 
protoplasts 0 

Nucleic acids (DNA, 

SiRNA, miRNA) 

Regeneration is highly inefficient for most 
species in transient studies and requires 
tissue culture 


mediated delivery 

Can lead to apoptosis and necrosis, 
random integration 

Narrow range of plant 
species, especially 
restricted from monooots 0 / 
mature plants, immature 
tissue, protoplasts 

Limited to DNA, large 
cargo size 

Leaf-targeted delivery is transient and gene 
edits are not transmitted to progeny, but allow 
diverse biological studies; requires tissue 
culture (except Arabidopsis) to generate 
progeny; exhibits high host-speoificity 

Viral delivery 

Virus integration (can be mitigated by 
using nonintegrating viruses) 

Host plant species 
restriotions/mature plants, 

Nucleic acids (DNA, 

SiRNA, miRNA), very 
limited cargo size 

Highly limited cargo-carrying capacity 

“While most biomolecule delivery methods to plants can deliver a variety of gene editing reagents, DNA plasmids are arguably the most common cargo of interest; DNA loading capacities are a useful metric 
for the upper limit for cargo sizes each method can sustain. 

b While biolistio particle-mediated delivery can theoretically be utilized in unlimited target species, the ability to target species depends on the target tissue (by extension, cell wall structural strength) and 
capability of available equipment. 

“The use of protoplasts as target tissue necessitates regeneration protocols and progeny segregation that are time-consuming and are challenged by the limited plant species amenable to protoplast 

d Progress has been made on increasing transformation efficiency in recalcitrant monooots [9]. 

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Box 2. Nanoparticles 

Nanoparticles (1-100 nm in at least one dimension) can be engineered with varied compositions, morphologies, sizes, 
and charges, enabling tunable physical and chemical properties. Ranging from zero to three dimensional, NPs are novel 
tools that have a wide range of applications, including but not limited to energy storage, sensing devices, and biomedical 
applications [109,110]. 

In addition to their high degree of tunability, NPs possess several advantages that validate their recent widespread use, 
with particular emphasis in the biomedical industry. Most NPs can be prepared with consistent properties for low batch- 
to-batoh variability, and can be designed to target biological systems, tissues, cells, or subcellular structures with high 
specificity [52], Moreover, NP-mediated gene and drug delivery can overcome common issues faced with viral vectors; 
NPs are often less immunogenic and oncogenic and can carry diverse and larger cargo, although the increased NP 
sizes when biomolecules are surface-loaded raise the challenge of bypassing biological barriers [111], Furthermore, the 
effects of NP use have yet to be thoroughly studied, though existing research points to nanoparticle chemistry, size, and 
dose as tunable parameters to control cytotoxioity[1 12,113], 

NPs are typically classified based on morphology and chemical properties. The most common categories include 
polymeric [114], lipid [115], magnetic [116], metallic [117], and carbon-based NPs [118], NPs can be synthesized with 
either a top-down or bottom-up approach using techniques such as lithography [119], deposition [120], and self- 
assembly [121], 

In NP-based delivery, a variety of strategies are employed to load NPs with the desired cargo. Physical techniques such 
as encapsulation or entrapment are commonly used in drug delivery to ensure the progressive release of drugs. 
Chemical techniques where the NP surface is modified for cargo grafting are in development, including noncovalent 
conjugation (electrostatic interaction [122], ir-ir stacking [123]) and covalent conjugation [23]. 

superior performance over conventional methods. For instance, Torney and colleagues’ MSN 
study achieved transgene expression with 1000 x less DNA than the tens to hundreds of 
micrograms of DNA typically required for conventional PEG-transfection in protoplasts [45]. 

A few recent examples show promise for NP-mediated passive delivery to plants in vitro [64-66] 
and in vivo [51,67] in, for example, N. tabacum protoplasts [66] and Arabidopsis thaliana roots 
[51,67], respectively (Table 3). Demirer and colleagues have recently achieved passive delivery 
of DNA plasmids and protected siRNA using functionalized CNT NPs for transient GFP 
expression in Eruca sativa (arugula) leaves and transient silencing of constitutively expressed 
GFP in transgenic Nicotiana benthamiana leaves [68]. This study also demonstrates CNT- 
mediated transient GFP expression in Triticum aestivum (wheat), indicating the potential for 
passive NP delivery in both model and crop species with high efficiency and low toxicity. While 
many more studies are needed to optimize NP properties and functionalization, these early 
results are promising for further exploration of NPs as a plant biomolecule delivery platform that 
addresses the shortcomings of conventional methods. Furthermore, with the advent of nucle¬ 
ase-based gene editing technologies (Box 3), it is of great interest to optimize the delivery of 
these revolutionary genome engineering tools by exploring NP-based delivery strategies for 
diverse biomolecular cargoes. 

Genome Editing has Enabled a New Era of Plant Science 

Engineered Nucleases for Plant Genome Editing 

Engineered nuclease systems, namely ZFNs, TALENs, and CRISPR-Cas, have emerged as 
breakthrough genome editing tools owing to their high genetic engineering specificity and 
efficiency (Box 3), whereby CRISPR-Cas has demonstrated increased simplicity, affordability, 
and multiplexing capabilities over TALENs and ZFNs in plants [69,70]. Since 2012, CRISPR-Cas 
has shown success for genome editing in both model and crop species, including A. thaliana, N. 
benthamiana, N. tabacum (tobacco), Oryza sativa (rice), T. aestivum (wheat), Zea mays (corn), 
Solanum lycopersicum (tomato), and Sorghum bicolor, among others [71 ,72]. Notably, CRISPR- 
Cas mutations as small as 1 bp have been conserved through three plant generations [73,74], 

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890 Trends in Biotechnology, September 2018, Vol. 36, No. 9 

Table 2. Challenges in Plant Genetic Engineering and Proposed Advantages of NP Delivery 

Desired outcome 

Nonheritable a 

(somatic/transient expression) 


(germline/stable transformation) 

Targeted tissue 



Protoplasts Zygotic embryo 

Somatic embryogenic calli 

Tissue-specific biological and 
experimental challenges 

• Cell wall 

• Inefficient cellular uptake 

• Epidermal barrier 

• Cell wall 

• Inefficient cellular uptake 

• Cell wall degradation 

• Inefficient cellular uptake 

• Cell wall 

• Inefficient cellular uptake 

• Embryo collection/calli 

• Calli regeneration 

• Cell wall 

• Inefficient cellular uptake 

• Totipotency/calli induction 

• Calli regeneration 

Proposed advantages of NP 

• NP-cell wall permeability 

• NP-stomata permeability 

• Anionic NPs root-to-shoot 
vascular translocation [46] 

• Passive uptake or direct 
mesophyll injection 
without gene gun or 

• Tunable NP properties 
and ligands for subcellular 

• NP-cell wall permeability 

• Cationic NP root 
accumulation [46] 

• Passive uptake without 
gene gun or protoplasts 

• Tunable NP properties 
and ligands for subcellular 

• Tunable NP properties 
and ligands for subcellular 

• NP-oell wall permeability 

• Tunable NP properties 
and ligands for subcellular 

• NP-cell wall permeability 

• Tunable NP properties 
and ligands for subcellular 

a While these somatic tissues (leaves, roots, protoplasts) are most commonly targeted for transient expression experiments, heritable outcomes may be derived through somatic embryogenesis 
(dedifferentiation of somatic tissue). 

Trends in Biotechnology CGlPrGSS 


Trends in Biotechnology, September 2018, Vol. 36, No. 9 891 

Table 3. Select Summary of NP-Mediated Genetic Engineering in Plants 

NP type 


Plant species; cell/tissue type 

Delivery method 




With external aid 

Gold capped MSNs 

GFP plasmid; chemical 
expression inducer 

N. tabacum cotyledons; Z. 
mays embryos 


Co-delivery and controlled release of 
DNA and chemicals 



Poly-L-lysine coated 
starch NPs 

GFP plasmid 

Dioscorea zingiberensis C.H. 
Wright calli suspension 


5% transient expression efficiency; 
some integration occurs 



Gold-plated MSNs 

GFP and mCherry plasmids; 
GFP protein 

Allium cepa epidermis tissue 


DNA and protein co-delivery 



Magnetic gold NPs 

(3-glucuronidase (GUS) 

Brassica napus protoplasts 
and walled cell suspension 

Magnetic field 

Transient GUS expression 



Gold-plated MSNs 

AmCyanl and DsFted2 
plasmids; Cre protein 

Z. mays embryos 


DNA and protein co-delivery; both 
transient and stable expression 



Dimethyl aminoethyl 
methacrylate (DMAEM) 
polymer NPs 

Yellow fluorescent protein 
(YFP) and GFP plasmids 

N. tabacum and Ceratodon 
purpureus protoplasts 

PEG transfection 

Both transient and stable expression 



Magnetic Fe 3 0 4 NPs 

Selectable marker gene 

Gossypium hirsutum pollen 

Magnetic field 

~1 % efficiency for generating stable 
transgenic seeds 



In vitro without external aid 

(PAMAM) dendrimer NPs 

GFP plasmid 

Agrostis stolonifera L. calli 


48.5% cells showed transient 



Calcium phosphate NPs 

GUS plasmid 

Brassica juncea hypocotyl 



80.7% stable transformation 



functionalized CNTs 

YFP plasmid 

N. tabacum protoplasts and 
leaf explants 


Both transient and stable expression 



In vivo without external aid 

functionalized MSNs 

mCherry plasmid 

A. thaliana roots 


46.5% transient expression efficiency 



PAMAM dendrimer NPs 

Double-stranded DNA for 

RNA interference 

A. thaliana roots 


Developmental gene silencing led to 
systemic phenotypes 



Polymer functionalized 

GFP plasmid; siRNA for 
transgenic GFP silencing 

E. sativa, N. benthamiana, 

and T. aestivum leaves 


95% transient silencing efficiency; 
transient expression in mature leaves 


Trends in Biotechnology CGlPrGSS 


Trends in Biotechnology 



Box 3. Traditional Genetic Engineering versus Nuclease-Enabled Genome Editing 
Genetic engineering refers broadly to manipulating a cell’s genome and gene expression profile. Techniques for genetic 
engineering may cause recombinant protein expression, up/downregulation of a gene, permanent gene knockout, 
targeted mutations in the host gene, or insertion of large foreign DNA segments into the host genome. Genome 
modifications may be transient, permanent, or heritable and involve many types of biomolecules (most commonly RNA, 
DNA, and proteins) which are sometimes taken up passively by cells but often require enhanced delivery techniques, 
such as gene guns, microinjection, electroporation, sonoporation, nanoparticle-assisted delivery, and engineered 
bacteria or viruses. In plants, genetic engineering is hindered by the cell wall, requiring delivery methods that are highly 
host-specific or limited by challenges in plant regeneration. 

Nuclease-enabled genome editing refers to techniques where genes are removed or changed with engineered 
nucleases, a class of enzymes that perform targeted double-stranded breaks (DSBs) at specific locations in the host 
genome. When nucleases perform DSBs, the cell undergoes homology-directed repair (HDR) or nonhomologous end¬ 
joining (NHEJ) to repair the cut. NHEJ is a random, error-prone repair process that involves realignment of a few bases, 
such that the high error frequency provides a simplistic pathway for gene knockout. HDR is a nonrandom repair process 
requiring large stretches of sequence homology, allowing for precise edits by introducing customized homologous 
recombination sequences for gene knockout, knock-in, and targeted mutations. Prominent tools in genome editing are 
zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly 
interspaced short palindromic repeat)-Cas (CRISPR associated) systems. In the 1990s, ZFNs became the first nuclease 
system engineered for selectable genome editing in bacteria |124], TALEN and CRISPR-Cas genome editing systems 
were developed for bacteria and eukaryotes more recently, around 2009 and 2012, respectively [125-128]. Composed 
of protein complexes containing a DNA-binding domain and a DNA-cleaving domain, ZFNs and TALENs rely on protein/ 
DNA recognition to induce endogenous DNA repair. CRISPR-Cas systems are composed of a nuclease protein (Cas) 
and a guide RNA (gRNA) with sequence homology to the genomic target, and therefore rely on the formation of a 
ribonucleoprotein (RNP) complex to induce HDR or NHEJ. While all three systems have their drawbacks, CRISPR-Cas 
has revolutionized the field of genome editing owing to its relatively superior simplicity, efficiency, and multiplexing ability 
(i.e., simultaneous editing of different genes) over ZFNs and TALENs. 

which is promising for stable transgene-free modified crops. As with traditional genetic engineer¬ 
ing of plants, many of the limitations for implementing gene editing tools in plants (low editing 
efficiency, tissue damage, species limitations, cargo-type limitations) originate in biomolecular 
transport into plant cells. As such, NP-based biomolecule delivery to plants stands to enable 
higher-throughput plant genome editing via DNA, single guide RNA (sgRNA), and RNP delivery, 
and thus warrants a discussion on the state of the plant genome editing field. 

Global Landscape of Regulatory Uncertainty towards Genetically Engineered Crops 
Genetic engineering of crops has evolved to overcome limitations in traditional breeding, as 
breeding is slow, laborious, and lacks precise control over plant genotype and phenotype 
generation. Modern biotechnology enables rapid development of crop variants with disease 
and pest resistance, stress tolerance, higher yield, and enhanced nutritional value. Since 1996, 
global genetically modified organism (GMO) cultivation has increased 110-fold to 185 mega¬ 
hectares in 2016 [75] (Figure 2). The US is a leader in GMO production but highly regulates 
production of modified crops, which poses, among other challenges, significant financial barriers 
to commercialization of new crop variants [76]. The US GMO pipeline is product-based but 
sensitive to plant pests, such that Agrobacterium automatically triggers regulation, while other 
methods of gene delivery are often deregulated if the product is nontransgenic [76,77]. European 
Union GMO regulation is process-based and affects any organism whose genome has been 
modified other than by mating or natural recombination [78], but includes exceptions for certain 
types of mutagenesis that will likely exempt modern gene editing [79], The advent of nuclease- 
based gene editing (Box 3) has set forth a global reevaluation of the legislation surrounding 
genetically engineered crops, wherein several leading GMO cultivators have exempted non¬ 
transgenic genome-edited plants from regulation (Figure 2). Recently, the USDA officially stated 
that there are no future plans to include genome-edited plants under the current US regulatory 
umbrellafor GMOs [131]. However, due to differences in regulatory philosophy and public opinion, 
several countries oppose deregulation of nontransgenic genome-edited plants and it remains 

892 Trends in Biotechnology, September 2018, Vol. 36, No. 9 

Trends in Biotechnology 



Area of GMO cultivation worldwide in 2016 (millions of hectares) 3 










Strict policy in Europe: 'Opt-out' model allows EU member states prohibit 
cultivation of EU-approved GMOs within their own territory. Most states opt-out GMO 
cultivation but allow imports for animal feed. 

Genome editing could bypass strict policy: In January 2018, European Court of 
Justice Advocate General states that plant mutagenesis by modern gene editing 
techniques may qualify for regulatory exemption in the EU. b 

South Africa Ml.? 
Uruguay! 1.3 
Bolivia 11.2 
Australia 10.9 
Philippines! 0.8 
Myanmarl 0.3 
Spainl 0.1 
Sudani 0.1 
Mexicol 0.1 

Nontransgenic genome edited plants are: 

0 Subject to GMO regulation 
# Not subject to GMO regulation 
0 Currently undergoing regulatory review 
0 Not yet explicitly addressed 

Sweden: CRISPR/Cas edited A. thaliana granted non-GMO status in 2015. c 

Regulatory review underway in China: Chinese 
government strongly supports GM crops, National 
Biosafety Committee still developing regulations for 
plant genome editing as of january 2018. d 

Canadian regulations differ 
from the rest: Canadian law 
addresses 'plants with novel 
traits' (PNTs) rather than GMOs, 
existing legislation adequately 
xovers plant genome editing! 

U.S. GMO regulation and pipeline cost: 6+ 

years, $50 million+ regulatory pipelines for GMOs, 
as many as 10 nuclease-edited plants bypassed 
regulation in the US as of January 2018. C 'S 

Argenetina pioneers plant gene editing 
legislation: In 2017, Argentina passed the first 
legislation specific to modern genome editing; 
nontransgenic gene edited plants are exempt from 
regulation^ As of 2018, similar resolutions were 
V^passed in Chile and Brazil!-' 

• Active GMO cultivation/trials 
0 Not restrictive to GMOs 

• Restrictive to GMOs 
No data available 

Regulatory review coming to an end in 
Australia: As of january 2018, following 
12-month review, Australian policy expected 
to loosen up for gene editing in plants. e 

Trends in Biotechnology 

Figure 2. Genetically Modified Organism (GMO) Cultivation and Regulatory Attitudes Worldwide. Despite a long, expensive regulatory pipeline, the US is a 
leader for GMO cultivation worldwide, followed by Brazil and Argentina, with Argentina being the first to directly address modern genome editing techniques in GMO 
legislation. European and Australian regulatory attitudes are strict but have recently evolved as of January 2018, suggesting that regulations for genome-edited plants 
will soon be relaxed in these regions. Nuclease-based edits without transgene integration escape regulation, even in countries with large agricultural GMO industries and 
complex regulatory systems. Globally, GMO regulation and commercial use is heterogenous and uncertain due to economic, ecological, and sociopolitical 
complexities. This map is a simplification of the convoluted global landscape regarding genetically engineered crops. ‘Restrictive to GMOs’ indicates a complete 
or partial ban on GMOs and GMO-derived products for commercial or research purposes. a [75], b [79], °[80] , d [1 03], e [104], *[105], 9 [106], h [132], '[133]. 

unclear how enforcement of GMO status will proceed worldwide in the future [80]. Despite the 
heterogenous and dynamic global regulatory landscape, nuclease-based genome editing cur¬ 
rently plays a critical role in overcoming regulatory restrictions and ensuring scientific progress, as 
well as commercial implementation of engineered crop variants. 

Nanocarriers Hold Promise for Nuclease-Based Plant Genome Editing 
Genome editing tools may increase the throughput of plant molecular biology and genetic studies, 
and as such could shift the paradigm in regulatory oversight of transgenic plants. Species, 
amenable tissue, expression strategy (DNA, RNA, or protein), and delivery method contribute 

Trends in Biotechnology, September 2018, Voi. 36, No. 9 893 

Trends in Biotechnology 



to the efficacy of transgene expression or modification and to the propensity of transgene 
integration into the host genome. ‘DNA-free’ genome editing techniques are increasingly attrac¬ 
tive, especially from a regulatory perspective, to eliminate all risk of transgene integration. Recently, 
RNP delivery has been demonstrated in A. thaliana and O. sativa protoplasts via PEG-transfection 
[81 ] and Z. mays embryos via gene gun delivery [82] ; the methods used in both of these studies are 
primarily throughput-limited by challenges in progeny regeneration. The challenge to realizing 
efficient, stable gene editing in plants is twofold. First, plant germline cells cannot be transformed 
by any current method (with the exception of Arabidopsis floral dip [83]) and therefore progeny 
must be regenerated from embryogenic calli. Second, the cell wall imposes a rigid transport barrier 
to biomolecule delivery, such that conventional delivery in plants is either destructive and ineffi¬ 
cient, or host-specific. Thus, the foremost limitation for broad-scale implementation of plant 
genome editing originates from an inability to target germline cells, and the absence of an efficient 
and species-independent bio-cargo delivery strategy. While engineered nuclease systems have 
begun to reveal remarkable potential for the future of plant genome engineering, novel carriers are 
required to overcome the restrictions of conventional delivery methods, but could also begin to 
pave the way for efficient progeny regeneration or direct germline editing in plants. 

NPs have begun to facilitate and enhance genome editing through efficient and targeted 
delivery of plasmids, RNA, and RNPs [84], In mammalian cells, NPs are routinely used for 
efficient, direct cytosolic/nuclear delivery of Cas-RNPs in many cell types [85], and RNP delivery 
has been shown to greatly reduce off-target effects in comparison with plasmid-based CRISPR 
systems [84], However, in plants, the cell wall has hindered the development of an analogous 
system that can passively deliver genome editing cargo to mature plants and across species. 
Thus, there remains much potential for designing NP carriers with diverse cargo loading 
capabilities (DNA, RNA, proteins) and optimal geometry/chemistry to efficiently bypass the 
cell wall and membranes in dense plant tissues without external aid. Previous work [51,67,68] 
shows that some NP formulations are capable of passive internalization in planta with DNA, 
RNA, or protein cargo. These NP scaffolds, namely CNTs, MSNs, and polymeric NPs, should 
be further explored for delivering engineered nuclease systems to plants. 

Concluding Remarks and Future Perspectives 

Genetic engineering of plants has greatly accelerated scientific progress and paved the way for 
crop variants with improved growth characteristics, disease and pest resistance, environmental 
stress tolerance, and enhanced nutritional value. In parallel, advances in site-specific genome 
editing technologies have optimized the precision with which genetic engineering of organisms 
can be accomplished. However, conventional methods of plant genetic engineering and genome 
editing are limited in scope. This is primarily due to the cell wall that imposes a barrier to efficient 
delivery of biomolecules, which could potentially be overcome by NPs. Agrobacterium is a 
preferred method for plant genetic transformation, but is only effective in a limited range of host 
species and is an automatic trigger for regulatory oversight in the United States. Biolistic particle 
delivery and PEG-transfection are effective, host-independent transformation methods, but 
difficulties in regenerating healthy plant tissue and low-efficiency editing are severe drawbacks 
to their broad-scale and high-throughput implementation. NPs have recently emerged as a novel 
method of targeted biomolecule delivery in mammalian cells, especially for clinical applications. 
However, exploration of nanocarriers for biomolecule delivery in plants remains a nascent field, 
with much potential for the future of plant biotechnology and genome editing (see Outstanding 
Questions). Preliminary studies show that NPs with proper surface chemistry and physical 
properties analogous to those developed for animal systems are capable of delivering biomo¬ 
lecules to plants in vivo and in vitro with improvements over conventional methods. However, as of 
yet, most nanocarriers in plants still require assistance from conventional methods (i.e., gene gun), 

Outstanding Questions 

Are there nanoparticle varieties yet to 
be discovered for efficient biomolecule 
delivery in plants, or do we lack knowl¬ 
edge of, or control over, optimal nano¬ 
particle modifications for applications 
in plant systems? 

Can we narrow the current design 
space to a single nanoparticle type 
with tunable functionalization for pas¬ 
sive delivery in plants, regardless of 
cargo type, plant species, and tissue 

How might we gain a better mechanis¬ 
tic understanding of nanoparticle inter¬ 
nalization into plant cells, and how can 
we harness this knowledge towards 
rational design of nanoparticles for a 
range of biological delivery 

Will challenges in biomolecule delivery 
and progeny regeneration always 
remain decoupled, or will nanoparticle 
delivery enable significant increase in 
throughput and efficiency of genetic 
studies on plant regenerative biology 
and stable transformation? 

While genome editing by induced non- 
homologous end-joining does not 
invoke regulatory oversight in many 
countries, how will genome edits intro¬ 
duced by homology-directed repair 
(where integration of a repair template 
is necessary) be classified from a leg¬ 
islative standpoint? 

How can scientists, the public, and 
regulatory bodies create a space for 
open communication to address the 
risks of introducing crop variants to 
the environment, while continuing to 
enable scientific progress and com¬ 
mercialization of sustainable and resil¬ 
ient crop variants? 

894 Trends in Biotechnology. September 2018, Vol. 36, No. 9 

Trends in Biotechnology 



or are limited to in vitro studies. To our knowledge, the field of plant bioengineering has yet to fully 
demonstrate a reliable strategy for NP-mediated passive biomolecule delivery to plants. To realize 
the full scientific and humanitarian potential in genetic engineering of both model and crop species, 
especially with the advent of nuclease-based genome editing, a promising focus will be to optimize 
NPs as efficient and ubiquitous delivery vessels of diverse biomolecules, tunable across cargo 
types, species, and tissues, for both transient and stable genetic engineering. However, because 
germline transformation is currently limited to only one model plant species (Arabidopsis), even a 
ubiquitous delivery strategy for precise genome editing would be limited by the success of 
regenerating progeny from somatic tissue. A remarkable, yet conceivable, future accomplishment 
of NP delivery in plants could be enablement of unprecedented, highly parallel genetic studies that 
elucidate the precedents for success in tissue regeneration, and the direct manipulation of 
germline plant cells. 


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