1 Introduction

Increasing global food security triggered by man-made and natural causes has been exerting tremendous pressure on the agricultural sector. The past century was witness to numerous technological innovations, such as the development of hybrid varieties, systemic development, and use of highly efficient synthetic chemicals that helped the growth in crop production (Ray et al. 2013; Fischer et al. 2014). However, a major boost was seen with the advent of biotechnology, which propelled the production of crops that were resistant to various biotic and abiotic factors including viruses, pests, drought, salinity, etc. (Altman and Hasegawa 2011). Though all this assisted greatly in addressing the global food management, numerous factors, natural and man-made, have been stalling the progress or alternatively creating sudden and excessive demands, varying by geographic locations, which have been posing new challenges for this sector (FAO 2018). Therefore, rises the need for a more advanced technological system for alleviating the problems faced by the agricultural community which can work in conjunction with the old system (conventional) and independently on its own.

Due to the various revolutionary applications in fields as diverse as medicine, materials science, electronics, energy, etc., nanotechnology has emerged as an indispensable asset in the modern world (De et al. 2014). It is only befitting that it is accredited as one of the six “Key Enabling Technologies” by the European Commission (European Commission Communication 2009). Investigations on the applications of nanotechnology in agriculture pertaining to sustainability, resistant/tolerant varieties, increased productivity, post-processing, packaging, and distribution, etc. have been going on for many years, but has only over the past decade began to take prominence, which is evident from the increased number of scientific publications and patents in this field (Rodrigues et al. 2017; Lowry et al. 2019; Mohamed and Kumar 2016a, b). The essence of nanotechnology lies in minimizing the essentials and delivering them to exactly the desired location with high precision, thus greatly reducing costs, decreasing undesirable wastage of resources, limiting offsite interactions, and having explicit and increased effects (Prasad et al. 2017). In agriculture, this pertains to significantly reducing the application of chemicals, instead employing a smarter delivery mode of active ingredients facilitating better and efficient essential resource (water, nutrients, etc.) management (Zhao et al. 2018). A more exciting and enticing application prospect of nanotechnology is in the field of plant breeding and genetic transformation (Elemike et al. 2019; Cunningham et al. 2018).

Genetic improvements of crops generally involve the cellular level diversion of genes to a desired location for target gene expression, which could be for a variety of purposes like imparting resistance to pest(s), tolerance to climatic anomalies or to induce additional productivity traits. These genetic alterations are intended for improving the overall quality of crops (Abdallah et al. 2015). A general routine of two distinct steps are followed during genetic engineering of plants: (1) transient transformation—the introduction of desired DNA into plant cells, and (2) stable transformation—subsequent integration of foreign DNA with plant genome. Though each step poses its own challenges, the proper integration of the transgene into the host genome is the most crucial part to produce transgenic plants with desired traits (Bates et al. 1990). This process of transformation is generally conducted under the protective environment of sterile laboratory conditions and engineered plants need to regenerate from transformed tissues which again introduces newer challenges.

Introduction or transfer of genes has been conventionally achieved by (1) natural method—using natural agents as Agrobacterium species, viruses, etc., (2) direct method—protoplast fusion, transfection, etc., (3) physical method—electroporation, particle bombardment, microinjection, etc., and (4) chemical method—gene transfer using plasma membrane destabilizing/precipitating agents as PEG, PVA, NaNO3, DEAE, dextran, DMSO, etc. (Demirer and Landry 2017). Although successful, these conventional methods were plagued with limited transformation rates due to various reasons, the most prominent being utilization of excessive energy and chemicals inflicting damage to DNA and cells. Circumventing these issues would result in an efficient process and a more productive outcome.

Nanotechnology-based processes promise effective solutions for such limitations. Various nanocarriers such as liposomes, polymeric and inorganic nanoparticles (NPs), carbon nanotubes (CNTs), and quantum dots (QDs) have been successfully tested for their abilities to carry genes of interest to the desired host system with commendable success rates (Cunningham et al. 2018). This chapter will dwell into the implications (past, present, and future) of nanotechnological interventions in the field of plant genetic engineering while comparing the conventional modules as well.

2 Conventional Plant Biotransformation Techniques

Methods for plant biotransformation have been adapting to the various advancements in time to achieve better and stable gene expression outcomes. Though technological advancements have been implemented, conventional gene delivery approaches are still being used (Cardi et al. 2017). These conventional methodologies fall under three predominant classes: physical (biolistic particle bombardment/gene gun, electroporation—using electric field pulses to create pores in cell membranes and microinjection), chemical (PEG-mediated), or biological (Agrobacterium-mediated) with their own advantages and limitations specific to the mode of application (Table 1.1). Yet, the limitations outweigh the advantages and hence the need for promising alternatives. Nanotechnological interventions that are being developed to circumvent these limitations of conventional approaches and simultaneously enhancing overall process efficiency are discussed in this chapter.

Table 1.1 Conventional gene delivery methods, advantages, and drawbacks
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3 Why Nanotechnology?

With conventional plant gene delivery methods facing serious issues, prospective alternatives that are cost-effective, easy, robust, non-toxic, and that can transfer genes into all phenotypes irrespective of the species become a necessity. Nanotechnology promises augmentation of agricultural productivity by highly specific and reliable genetic transformation methods at the cellular level (Sekhon 2014).

The nano-approach advertises numerous advantages over the conventional practices, with regards to its ease of operation and high efficiency (Mishra et al. 2017). For example, a thousand times less amount of DNA is required for the detection of expression than the conventional methods. Transient DNA-free genome editing (including gene silencing) is enabled in a controlled fashion. Co-delivery of multiple biomolecules (DNA activator, DNA proteins, different genes), simultaneously to the target, is facilitated. The surface of the gene nanocarriers can be easily functionalized using certain bio-recognition entities for specific, targeted delivery. The non-toxic nature of nanocarriers and their local translocation to individual cells, organs, or tissues is an additional advantage.

Particles with size ranging from 1 to 1000 nm are regarded as nanoparticles or nanocarriers (Nair et al. 2010, 2012). The distinctive small size and unique surface properties project them as versatile carriers for genes and other biomolecules. NP-based biotransformation proposes numerous benefits: (i) faster gene transfer than Agrobacterium-based process, (ii) DNA encapsulated in protective layers therefore resistant to damage/inactivation, (iii) gene mutation due to energy-induced DNA damage (typically in physical methods) is greatly reduced, (iv) greater control over transformation process, and (v) amalgamation of nano- and physical methods is possible leading to effective and improved gene transfer. So far, a wide range of nanomaterials such as liposomes, CNTs, QDs, and silica NPs have been used for plant gene engineering (Nair et al. 2010).

In contrast to the animal system where the application of nanotechnology for gene delivery has been widely demonstrated, in plants, owing to the presence of a rigid cell wall which restricts the transport of molecules into plant cells, research has been slightly sparse (Rai et al. 2012). NPs have interesting value as intracellular biomolecule delivery materials, such as (i) their biological membranes trespassing ability, (ii) ability to carry, shield and release various payloads, and (iii) attain specific targeting by facile surface modification (Sokolova and Epple 2008). The NP-based gene delivery in plants is limited not only by the cell wall but also by the general in vitro plant studies. The regeneration capacity in in vitro cultures varies greatly across species, genotype, and even in individual plant depending on the developmental age of source tissue. As of now, regardless of the delivery method, all stable transformations need progeny regeneration from embryogenic calli. Therefore, delivery and regeneration optimizations are essential for improving the efficiency of stable transformation across a wide range of plant species.

4 Nanocarriers for Plant Biotransformation

NPs for the delivery of biomolecules (nucleotides, activators, proteins) to plants in vitro and in vivo (Mohamed and Kumar 2016a) are being explored at a much-enhanced pace than ever before. NP-mediated genome editing to deliver green fluorescent protein (GFP) gene-containing plasmid and functional recombinase into plant tissues has been a great success. One prominent example is that of mesoporous silica NPs (MSNs) for delivery of DNA and its activator into tobacco plant via isolated cells and intact leaves (Torney et al. 2007). Furthermore, MSNs, single-/multi-walled CNTs (SWCNTs/MWCNTs), gold NPs (AuNPs), magnetic virus-like NPs (MVNPs), carbon-coated magnetic NPs, QDs, and starch NPs have been employed in this regard (Mohamed and Kumar 2016b; Nair et al. 2012).

4.1 Silica Nanoparticles

Looking at a few detailed examples, MSNs-DNA complex was delivered into intact roots of Arabidopsis thaliana without any involvement of mechanical force (Chang et al. 2013). Fluorescence labeling along with antibody detection was used to confirm the gene expression from the epidermal and inner cortical and endodermal (not possible with conventional physical methods) root tissues. Simple co-culture was enough for MSNs to infiltrate the cell wall resulting in transient gene expression in intact Arabidopsis roots, thereby negating the need for physical injury causing conventional biolistic approaches (Fig. 1.1).

Fig. 1.1
figure 1

Reproduced with permission from RSC

MSN-mediated gene delivery. a, b Confocal microscopic images of DNA-MSN complex (1: 100 ratio) treated (48 h at 24 °C in 1/2 MS) Arabidopsis root cells. Gene expression (mCherry protein; red) was observed in endodermal (a) and cortical (b) cells. TMAPS/F-MSNs were present in cells expressing mCherry (b, green channel). Scale bars: 50 mm. c, d TEM of immunogold-labeled mCherry protein in root cells after incubation with DNA-MSN complexes. Red arrows show the gold-labeled mCherry proteins. Presence of TMAPS/F-MSNs (black arrow) and mCherry protein (red arrows) in the same cell (d). Scale bars are 200 nm. Cp, cytoplasm; M, mitochondrion; V, vacuole; G, Golgi apparatus.

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Biolistics mediated co-delivery of AuNP functionalized MSNs (Au-MSN), with large average pore diameters (10 nm) and fluorescently labeled protein, bovine serum albumin (BSA) along with plasmid DNA, an enhanced green fluorescent protein (eGFP) into Allium sativum (onion) plant tissues was reported (Ortigosa et al. 2012). A proof-of-concept experiment demonstrated Au-MSN delivery and release of proteins and plasmid DNA in the same cell after trespassing the cell wall via biolistic approach. This was the first example of co-delivery of active non-denatured proteins and plasmid DNA (eGFP fluorescence) to plant cells providing new insights into NP-mediated cargo delivery to plants.

Genome editing can be carried out without direct delivery of DNA as such, rather with proteins or enzymes that can initiate/facilitate biochemical analysis or genome modifications. This can negate DNA (transgene)-genome integration and result in precisely modified “non-transgenic” plants. In this regard, gold-plated MSNs-protein (Cre recombinase) complex were used to deliver the protein via biolistics into Zea mays (maize) cells harboring loxP sites flanking a selection gene and a reporter gene (Ortigosa et al. 2014). With the release of Cre protein from MSNs, recombination of loxP sites followed by elimination of both genes occurred. Fertile plants were regenerated from selected recombination events with nearly 20% of embryos developing calli with recombined loxP sites. This technology is a DNA-free module for genome editing where MSNs can be modified to accommodate specific enzymes and target desired tissue.

Torney and co-workers utilized honey-comb-like MSNs to transport DNA and chemicals into Nicotiana tabacum via biolistics (Torney et al. 2007). MSNs were loaded with the gene and its inducer, and to further prevent unwanted escape from MSN pores, the ends were capped with AuNPs through disulfide bonds. The significance of disulfide bonds is in cellular environments, where they are reduced, resulting in the release of DNA, conditions which can be precisely controlled. Optimal DNA/MSN ratio was found to be 1:10 (w/w), wherein, a stable DNA-MSN complex was formed, without any free DNA in solution post 2 h of incubation. Transient GFP expression was observed post 36 h of DNA-MSN incubation (Fig. 1.2). This stable nanoconstruct can be utilized for genetic transformation of plants or to induce pathogen-activated defense response.

Fig. 1.2
figure 2

Reproduced with permission from NPG

Confocal imaging of MSN uptake by tobacco mesophyll protoplasts. Protoplasts incubated with a–c, type-I MSNs (single focal plane images) and d–f, type-II MSNs (three-dimensional reconstruction images). No uptake of type-I MSNs was observed, but type-II MSNs were internalized. Both MSNs are functionalized with fluorescein and visible in green (thick arrows). Auto-fluorescing chloroplasts in the protoplasts are in red (thin arrows).

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4.2 Calcium-Based Nanoparticles

In another work, 20–50 nm calcium phosphate (CaP) NPs carrying a reporter gene (pCAMBIA1301) was employed (Naqvi et al. 2012). Gel electrophoresis was used to determine pCAMBIA1301 encapsulation and pH-dependent dissolution of CaPNPs. The encapsulation of DNA in CaPNPs was highly beneficial in protecting it from intracellular nucleases. A transformation efficiency of 80.7% was achieved with the nanocarrier when compared to Agrobacterium tumefaciens (54.4%) and naked DNA (only 8%), clearly highlighting the superiority of the NP-mediated genetic transformation over the conventional module. The authors suggest the release of plasmid DNA from CaPNPs in the cell, perhaps facilitating nuclear entry as opposed to the case of A. tumefaciens infection where transgenic GUS (b-glucuronidase) integrates into genomic DNA by non-homologous recombination. Also, this nanoconstruct did not cause any injury to the plant cell while delivery (Fig. 1.3).

Fig. 1.3
figure 3

Reproduced with permission from RSC

Confocal scanning laser microscopy of FITC–dextran loaded CaP nanoparticles incubated with hypocotyl section of Brassica juncea L. cv. Pusa Jaikisan at different time intervals, i.e., a 5 min, b 4 h, c 8 h, d 12 h, e 16 h, f 20 h, and g 24 h.

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In a similar kind of work, some researchers developed CaPNPs (25–55 nm) and successfully delivered pBI121 harboring GFP driven by 35S promoter-encoding plasmid DNA in tobacco plants (Roy et al. 2003). Due to pDNA being anionic, easy encapsulation into CaPNPs was achieved. The pBI121 with GFP-loaded CaPNPs was an alternative non-viral vector for easy and efficient gene delivery while providing protection from cellular nuclease enzymes.

It is known that gene encapsulation into biocompatible carriers augments the biotransformation process and provides protection to the gene, cell, and enzymes. Genetic manipulation of Cichorium intybus L. with CaPNPs loaded with pBinAR containing trans-hmgr gene was performed (Rafsanjani et al. 2016). A transformation efficiency of 9.6% was recorded, with the transgenic plants showing higher levels of chlorophyll, soluble protein and esculin, attributed to the higher expression of hmgr transgene and increased activity of HMGR enzyme. These nanoparticles could be useful in producing genetically engineered plants of desired and enhanced traits.

4.3 Magnetic Nanoparticles

Magnetic gold nanoparticles (mGNPs) covalently bound to fluorescein isothiocyanate (FITC) molecules were delivered into Brassica juncea (canola) cells with/without cell walls under an external magnetic field (Hao et al. 2013). The translocation of NPs was confirmed from sectional transmission electron microscopy (TEM) images and further by confocal images where green fluorescence in the protoplasts and walled cells was observed, with a delivery efficiency of 95%. Further, canola cells with/without cell walls were targeted with plasmids-mGNPs complex. Post 48 h culture, blue color (X-Gluc) developed in the protoplasts, indicating the expression of the GUS gene. Meanwhile, intact cells (with cell wall) exhibited a green color, possibly a mix of blue and yellow from the suspension cells themselves. The positive gene expression and non-cytotoxic nature of mGNPs even with the toxic dye FITC, project this nanocarrier as an efficient gene and biomolecule delivery vector.

It is a fact that majority of the genetic modification modules depend on tissue regeneration from culture, often associated with long and arduous processes. In addition, certain crop species pose difficulty in regeneration, for example, cotton. Therefore, a novel transformation technique, pollen magnetofection, was developed for the direct production of transgenic seeds, bypassing regeneration (Zhao et al. 2017). Herein, exogenous DNA-loaded magnetic nanoparticles (MNPs) were directly delivered into pollen under a magnetic field. These magnetofected pollen were utilized for pollination, resulting in transgenic plants from transformed seeds. The successful integration of exogenous DNA into the genome was expressed and inherited in the offspring. The advantages of such a system are that it does not require laborious culture procedures and is genotype independent. Additionally, it is a facile and rapid process with multi-gene transformation capabilities and suitable for almost all crops, with an imminent impact in developing new varieties of transgenic crops.

4.4 Dendrimers

Cationic polyamidoamine (PAMAM) dendrimers are nanoparticles with a tunable number of branches and unique molecular features, making them one of the most promising nanocarriers for gene delivery applications. A diethylenetriamine core hyperbranched PAMAM (hPAMAM)-G2 dendrimer was used to conjugate with DNA (ssDNA-FITC-hPAMAM) and tested with delivery to alfalfa cells. Additionally, the efficacy of ultrasound (US)-mediated delivery of hPAMAM-G2 and gene expression (gusA) was probed (Amani et al. 2018). The electrophoresis retardation analysis (DNA-dendriplex dissociation) portrayed partial DNA protection from sonication damage with N/P ratios (where N is the number of nitrogen atoms in dendrimer and P is the number of phosphorus atoms in DNA) < 2 and with better DNA protection corresponding to increasing N/P ratios. Sonication increased the ssDNA delivery by 36%, significantly higher than without sonication group. Also, the N/P ratio was essential in determining the efficiency of transfection and gusA gene expression with the highest efficiency (1.4%) recorded at an N/P ratio of 10. The US (120s) and hPAMAM–DNA combination proved to be optimal with the highest gusA gene transfection and expression of 3.86%.

A similar work involving supramolecular complexes of G4 poly(amidoamine) dendrimers with GFP-encoding plasmid DNA via electrostatic interaction, to Agrostis stolonifera L. (creeping bentgrass) was reported. The fluorescently (TRITC) labeled dendrimers were recorded to enter the cells of turfgrass and expressing the GFP gene in the plant cells as observed by confocal fluorescence microscopy. It was concluded that the efficiency of transfection might be further enhanced with optimization of conditions as cell culture media pH and the molar ratio (N/P) of dendrimer/DNA (Pasupathy et al. 2008).

4.5 Liposomes

Liposomes are fascinating particles which bear a close resemblance to the plasma membrane of cells, mostly synthesized by components found inside the living system, thereby rendering them high biocompatibility. Their flexibility and ability to carry large payloads garner them unprecedented scientific appreciation. Deshayes et al. developed negatively charged liposomes encapsulating an Escherichia coli plasmid, pLGV23neo, carrying a kanamycin resistance gene (Deshayes et al. 1985). PEG was used to facilitate the fusion of liposomes with tobacco mesophyll protoplasts. Kanamycin-resistant clones were isolated from transfected cultures at an average frequency of 4 × 10−5 with the leaf protoplasts being resistant to kanamycin at 100 µg/mL. Aminoglycoside 3′-phosphotransferase II, a pLGV23neo encoded enzyme, was detected in the leaf extracts. At least one of the transformants had approximate 3–5 copies of the kanamycin resistance encoding genes inserted in their genome, making the experiment a commendable success.

In another work, Caboche and co-workers prepared unilamellar liposomal vesicles encapsulating pUC8CaMVCAT plasmid vector with the CAT gene (chloramphenicol acetyl transferase) and successfully introduced the gene in tobacco protoplasts (Caboche and Lurquin 1987). Ballas et al. synthesized tobacco mosaic virus RNA (TMV RNA)encapsulated liposomes (formulation of phosphatidylcholine/cholesterol) (Ballas et al. 1988). The surface of the so-developed liposomes was functionalized by quaternary ammonium detergent, di-isobutyl cresoxyethoxyethyl dimethyl benzylammonium (DEBDA [OH–]), thereby introducing surface hydroxyl groups which facilitated efficient TMV RNA transfer into tobacco and petunia protoplasts, within 48 h.

Wiesman et al. synthesized liposomes utilizing vernonia oil (a natural epoxidized triglyceride)-derived cationic amphiphilic compounds for DNA encapsulation (Wiesman et al. 2007). Sawahel utilized lipofectin for effective transfusion of N. tabacum and N. plumbaginifolia mesophyll protoplasts (Sawahel 2002). During their experiments, they confirmed that the CaMV-35S promoter controlled ß-glucuronidase gene had lower transformation efficacy than the PEG-mediated method.

4.6 Gold Nanoparticles

Due to their unique properties at the nanoscale, gold nanoparticles have found extensive applications in sensors and other biomedical applications. Though their use in plant biotransformation has been limited, it is on a steady rise. AuNPs-embedded carbon matrices were synthesized by heat treatment of the intracellular biogenic AuNPs produced by the fungus Aspergillus ochraceus. Vijayakumar and co-workers utilized these AuNPs (5–25 nm) for the successful delivery of plasmid DNA into N. tabacum utilizing gene gun (Vijayakumar et al. 2010). A similar DNA delivery demonstration was conducted in the same work with Oryza sativa (a monocot) and Leucaena leucocephala (a hard dicot tree species) with negligible cellular damage. Compared with conventional gene gun approaches using micrometer Au particles, it was established that the small size of AuNPs assists in easy and efficient embedding in the matrices which in turn enhances the transformation efficiency. On the other hand, less raw material (gold and plasmid) is needed to achieve the same level of transformation efficiency, aided by lower toxicity levels as well.

DNA-coated AuNPs were used for stable transformation of commercially important cultivars of Glycine max L. (soybean) callus. Soybean embryos (immature) were targeted with highly accelerated DNA-NP complex. Subsequent protoplast cultures were initiated under controlled conditions for the introduced neomycin phosphotransferase II gene. Kanamycin-resistant calli were obtained from the transfected cultures. Foreign gene expression was confirmed with enzyme assays and Southern blot hybridization (Christou et al. 1988).

4.7 Carbon Nanotubes

Liu et al. (2009) were supposedly the first to exploit CNTs for delivery of genes to plants. The independent (externally unaided) cellular uptake of both SWCNT/FITC and SWCNT/ssDNA-FITC conjugates was demonstrated in N. tabacum cells, validating the CNTs ability to penetrate intact plant cell walls and cell membranes (Liu et al. 2009). Later, Giraldo et al. (2014) injected the ssDNA-SWCNTs into the underside of A. thaliana leaves, using a syringe. They found that certain CNTs (defined size and charge) penetrated cell membranes without any external assistance (gene gun). The near-infrared (NIR) fluorescence emission of SWCNTs was used to understand their transport and localization in plant cells. Leaf tissue cross sections revealed the localization of ssDNA-SWCNTs in the leaf lamina and veins in intra- and extracellular parenchyma tissues and in chloroplasts (Fig. 1.4). These observations were depictive of successful trespassing of the plant cell wall, cell membrane, as well as the chloroplast membrane. Curiously, the leaf life span and chlorophyll content seemed to be unaffected by the infiltrated ssDNA-SWCNT solution, an assurance of non-cytotoxic nature of the complex (Giraldo et al. 2014).

Fig. 1.4
figure 4

Reproduced with permission from NPG

Nanoparticle transport inside isolated chloroplasts and leaves. a CRi Maestro images of ss(AT)15–SWNTs within the leaf lamina of A. thaliana. b–d, Co-localization of ss(AT)15–SWNTs near leaf veins (b), in parenchyma cells (c), and chloroplasts in vivo (d).

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Demirer and co-workers recently utilized covalently functionalized or pristine SWCNTs and MWCNTs for an efficient DNA delivery and robust transient protein expression in mature Eruca sativa (arugula) leaves. In the same study, cell wall-free arugula protoplasts were used for CNT-based transient protein expression, with a transformation efficiency of 85%. Again, in the same study a remarkable 95% gene (GFP) silencing efficiency was recorded for CNT scaffold-siRNA complex in N. benthamiana plant cell cytosol. The group was the first to demonstrate the efficient transient gene expression and silencing in mature plants by the use of CNT-biomolecules complexes. The results could spur high-throughput plant genetic transformation applications. The same group developed an efficient diffusion-based delivery of biomolecules with pristine/chemically functionalized SWCNTs and MWCNTs into tissues/organs of intact plants of several species. In N. benthamiana (Nb), E. sativa (arugula), Triticum aestivum (wheat), and Gossypium hirsutum (cotton), efficient delivery of DNA followed by robust protein expression (devoid of transgene integration) was achieved. Additionally, a gene silencing efficiency of 95% was observed with small interfering RNA (siRNA) delivery to Nb leaves. Here again, the nanomaterials apart from ferrying biomolecules into plant cells were able to safeguard polynucleotides from nuclease degradation. The results encourage species-independent, passive delivery of biomolecules, devoid of transgene integration, for various biotechnological applications (Demirer et al. 2019).

4.8 Other Nanoparticles

ZnS nanoparticles (3–5 nm) modified with positively charged poly-L-lysine were utilized for transformation of tobacco cells by delivering GUS-encoding plasmid DNA (pBI121), assisted by ultrasound (Fu et al. 2012). Molecular analysis as polymerase chain reaction (PCR), Southern blot, and GUS histochemical staining was performed on regenerated plants. Higher transformation efficiencies (43.42, 48.57, and 52.67%) were recorded with ZnS NPs-DNA complex as compared to conventional US-based transformation route (30–40%). This is primarily attributed to the shielding effect provided to the DNA by ZnS NPs. The stable expression results by ZnS NPs demonstrate their role as gene protector and carriers (Fu et al. 2012).

Quantum dots have been widely used as fluorescent bio labels in in vitro cell and in vivo animal imaging studies and off late for theranostics (therapy and diagnostic) as well (Mohamed et al. 2016). In plant sciences, QDs are mainly utilized for tracking the fate of NPs-DNA complexes during/post cell transfection and subsequently in plant transformation. Water-soluble CdSe QDs were synthesized for labeling chitosan-DNA NPs via electrostatic interaction. This CdSe-chitosan-DNA complex was utilized for the genetic transformation of Jatropha curcas cells. Full-length PCR amplification of the reporter gene (GFP) revealed that the DNA was not degraded in J. curcas cells transfected with the nanocomplex rather the target DNA was integrated into the plant genome as evidenced with fluorescence detection of the complex in the transformed calli (Wang et al. 2011).

Virus resistance in plants can be imparted by the topical application of pathogen-specific double-stranded RNA (dsRNA), an attractive alternative to transgenic RNA interference (RNAi). However, spraying naked dsRNA onto plants exposes its instability, thereby limiting practical application. Mitter et al. loaded dsRNA in layered double hydroxide (LDH) clay nanosheets which were non-toxic as well as biodegradable. The advantages of such a carrier include stable and robust dsRNA loading that does not wash off, presents sustained release from the nanocarrier and can be detected even after 30 days of application from sprayed leaves. LDH degradation, dsRNA uptake by cells, and homologous RNA silencing were recorded (Fig. 1.5). On comparison of sprayed and newly emerged unsprayed leaves, a single spray of dsRNA-LDH complex proved to provide effective virus protection for at least 20 days. The results witnessed in this work translate to the near future development of RNAi-nanosprays for a diverse range of applications (Mitter et al. 2017).

Fig. 1.5
figure 5

Reproduced with permission from NPG

BioClay (dsRNA-LDH) spray provides protection against viruses in local lesion assays. a Local lesions caused by CMV inoculation on cowpea. Plants at the two-leaf stage were sprayed with LDH, CMV2b-dsRNA, and CMV2b-BioClay on day 0 (n = 8–16 leaves per treatment group). Plants were mechanically inoculated with CMV at 1- or 5-day post-treatment. Lesions were counted 10 days post viral challenge (pvc). b Local lesions caused by PMMoV inoculation on N. tabacum cv. Xanthi. Plants were sprayed with either water, LDH, PMMoVIR54-dsRNA, or PMMoVIR54-BioClay on day 0 (n = 10–25 leaves per treatment group). Plants were mechanically inoculated with PMMoV at either 5- or 20-day post-treatment and necrotic lesions were counted 10 days pvc. c, d Images are showing the extent of necrotic lesions on N. tabacum cv. Xanthi leaves challenged with PMMoV 5-day post-spray treatment (c), and 20-day post-spray treatment (d). *P < 0.05, **P < 0.01 and ***P < 0.001 are significant using the Kruskal–Wallis test with post-hoc Nemenyi test for multiple comparisons between samples compared with LDH. Data represent mean ± s.e.m.

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Starch, owing to its biocompatible and biodegradable nature, has been studied in the nanoform for gene delivery applications. The starch NPs-gene complex was efficient enough to cross the cell wall, via US-assisted induction of transient pores in the cell wall, cell/nuclear membrane. Working in the same area, Liu and co-workers synthesized poly-L-lysine coated starch NPs (50–100 nm) in water oil microemulsion and surface modified with fluorescent [Ru(bpy)3]2+·6H2O conjugated to pEGAD plasmid DNA. They demonstrate the DNA protective effects of starch NPs in the DNA-NP complex from US damage and DNase I cleavage in plant suspension cells of Dioscorea zingiberensis. It was concluded that starch NPs could successfully be used in plant transformation as efficient nanocarriers of nucleic acids, assisted by US (Liu 2008).

Apart from improving yield, imparting disease resistance via gene transformation is also an essential aspect of sustainable agriculture. Chitosan NPs were encapsulated with thionin genes (AT1G12660 and AT1G12663) from Arabidopsis thaliana, in pEGAD vector and delivered to potato plants (Abdel-Razik et al. 2017). Thionin is an antifungal protein with action against pathogenic fungi of potato as Alternaria alternata and Rhizoctonia solani. Two prominent potato cultivars (lady and spunta) were chosen for transformation studies. RT-PCR was used for confirmation of thionin transgene expression in transgenic plants. Two assays were performed on transgenic potatoes to test resistance to the pathogenic fungi, (i) spore suspension effect on potato organs and (ii) effect of thionin on spore germination inhibition. New transgenic cultivars were highly resistant to the fungal infection symptoms as compared to non-transgenic normal plants. These chitosan NPs-DNA complex can be an effective tool to augment disease/pest resistance in various crop species.

While further extensive research is required to fine-tune the properties and functionalization of nanoparticles, the current scenario seems promising for aggressive application of NP delivery platforms for biomolecules to plants, addressing the limitations of traditional methodologies. Biomolecular delivery to plants is marred with limitations as low gene-editing efficiency, tissue/organ damage, species limitations, cargo limitations, etc. As compared with traditional modules, NP-based biomolecule delivery promises higher-throughput plant genome editing via DNA, single-guide RNA (sgRNA), and RNP delivery, thus opening new avenues for exploration in this exciting field.

Plants have remained elusive to the advances of technology in genetic transformation techniques, especially due to the rigid cell wall hindering the delivery of essential gene modification capable cargo to mature plants. This curiously reveals a vast scope for potential in designing much more efficient nanocarriers with distinct cargo (nucleic acids, proteins) ferrying capabilities and optimal physicochemical features for efficient and independent bypass of the inherent barriers in intricate plant tissues.

5 Patented Technologies

Though limited, there has been a rise in patent applications in the field of nanocarrier-based biomolecule delivery to plants, especially with regard to enhancing specific traits of desired plants via transformation. Recent patents 20110065092A1 (gene-encapsulated non-viral particle) and US6534484 (gene-encapsulated liposome for biotransformation) highlight the importance and necessity of nanotechnology in biotransformation. Few more patents assigned to nanotechnology-based biotransformation are enlisted in Table 1.2.

Table 1.2 Few nanoparticle-based patents on plant biotransformation
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6 Concluding Remarks and Future Perspectives

Engineering of plants at the gene level has impacted the global society in an unimaginable manner and has accelerated scientific ingenuity to develop crop variants with enhanced features as resistance to pests and diseases, tolerance to stress (abiotic/biotic), and enrichment of nutritional values. Advanced site-specific genome editing technologies have fine-tuned the precision with which genetic augmentation of plants is accomplished. Conventional approaches are greatly limited by the presence of the rigid cell wall, which makes penetration of biomolecules near to impossible without significant damage or loss of action. Agrobacterium has been the most revered plant genetic transformation tool but has limited host species range and is consistently under regulatory oversight in many countries. Biolistic and PEG transfection, though boast some perks over their biological counterpart, face severe difficulties in healthy plant regeneration from transformed tissues with low-editing efficiency. NPs are now established in in vitro mammalian cultures with a large population finding its way to human trials and further clinical applications. On the other hand, they are still in infancy when it comes to application in plants with much scope for investigations in future plant biotechnology and genome engineering. Initial reports have shown the potential of properly synthesized and functionalized analogues that can deliver biomolecules to plants in vitro and in vivo with better efficiency than conventional methods, though still some of them require external aid (i.e., gene gun). Not only the nanocarriers, but the auxiliary systems such as the ultrasound and the magnetic field generation systems should also be developed to better support the nanocarriers in enhancing their inherent potentials to the extent possible. The steps to the future are happening now, with numerous researchers fully engrossed in finding the perfect nanosystem for delivering the most sensitive of biomolecules across the most rigid cell walls to achieve the best transient gene expression efficiency so far. Slow, but sure, nanotechnology with its vast trove of nanocarriers and delivery mechanisms is the hope for fighting the various agricultural issues plaguing this planet now.