In planta NANO-delivery of nucleic acids into wheat and other cereal crop seed

Abstract

A method of introducing a polynucleotide into wheat seed using nanoparticles, e.g., mesoporous silica nanoparticles or silica nanoparticles comprising a polynucleotide, e.g., a polynucleotide for use in gene editing, transgene insertion, upregulation of gene expression, or downregulation of gene expression.

Description

IN PLANTA NANO-DELIVERY OF NUCLEIC ACIDS INTO WHEAT AND OTHERCEREAL CROP SEEDCROSS REFERENCE TO RELATED APPLICATIONS(0001] ] This application claims priority io U.S. provisional patent application no. 63 / 664,298, which was filed J une 26, 2024, U ,S. provisional patent application no. 63 / 664,309, which was filed June 26, 2024, and U.S. provisional patent application no. 63 / 664,322, which was filed June 26, 2024, which is hereby incorporated by reference in their entirety.TECHNICAL FIELD(0002] The present disclosure relates to a method of introducing a polynucleotide into Triticum (wheat). Sorghum (sorghum), Zea (com), and other cereal crop seeds, such as in a genotypeindependent manner, using nanoparticles, such as mesoporous silica nanoparticles and silica nanoparticles comprising a polynucleotide, such as a polynucleotide for use in gene editing, upregulation of gene expression, or downregulation of gene expression.BACKGROUND

[0003] Plant genetic engineering, also called genetic transfonnation, plays a pivotal role in crop molecular improvement Scientists use this technique to introduce valuable traits to crop varieties, such as enhanced productivity, improved nutritional quality, increased tolerance to abiotic stresses, enhanced disease resistance, and herbicide resistance.

[0004] Compared to dicot species, monocot species, particularly cereal crops, tend to be extremely difficult to transform. In cereals, where the transfonnation frequency is notably low and the process is challenging, there is a keen interest in employing an effective and straightforward method for gene transfer and regeneration (O’ Kennedy et al., 2006a;Raghuwanshi & Birch, 2010; Williams et al., 2004). The limited ability-' to transform cereal crops like wheat is a major barrier to molecular breeding and the widespread adoption of biotechnology for trait development.

[0005] For wheat, the earliest report for transformation was from Monsanto in 1992 on genotype ‘ Bobwhite’ (Cheng et al., 1997) using microprojectile bombardment to create an herbicide- resistant wheat variety (Vasil et al., 1992). Improvement in transformation efficiency was reported by using “immature embryo” as an explant. Agrobacterium-mediated tissue culture wasused to transform the wheat variety ‘Fielder’ with a 40-90% transformation efficiency in Japan in 2015 (Ishida et al, 2015). The most important issue with these tissue culture-based methods is their genotype dependency, which means only a handful of varieties can be transformed, even though thousands of wheat varieties, which differ in their agricultural importance, exist. For example, the two most commonly used wheat varieties, which have been successfully transformed, are ‘Fielder’ and ‘'Bobwhite’ but neither one is currently commercially cultivated anywhere in the world. In addition, the very low transformation efficiency, if it works on rare varieties other than ‘Fielder’, and the amount of time to generate To plants, i.e., - 1 year, a re bottlenecks for the application of methods such as genome editing.[0006| For sorghum (Sorghum bicolor), given its recalcitrance to transformation, genetic improvement traditionally depends on conventional breeding methods, but its limited responsiveness to genetic manipulation in vitro hinders the use of genetic transformation for enhancement. Despite challenges in sorghum tissue culture and transformation, its recognized significance in food and industrial crops, especially in the face of climate change, emphasizes the need for focused efforts on sorghum crops to ensure sustainable food and nutritional security in the future (Chou et al, 2020; Grootboom et al., 2010; and O’Kennedy et al., 2006 b). Each technique used to deliver genes into a plant genome, such as Agrobacterium-mediated transformation, microinjection, particle bombardment polyethylene glycol (PEG)-mediated transfection, and viral transfection, has inherent limitations, including high expenses, low effectiveness, and / or complex protocols. Notably, each cell tends to be responsive to only one or a few specific methods (Altpeter et al., 2005a; Delporte et al., 2014a; and Kooecianska & Wypijewski, n.d.). Besides strong genotype dependence, highly technical requirements of these systems limit their broad application (Altpeter et al., 2005a; and Delporte et al., 2014a). In various cereals, including sorghum (Sorghum bicolor), where the transformation frequency is notably low and the process is challenging, there is a keen interest in employing an effective and straightfonvard method for gene transfer and regeneration (O’Kennedy et al.. 2006a;Raghuwanshi & Birch, 2010; and Williams et al., 2004). The limited ability to transform sorghum is the major barrier to the widespread adoption of sorghum as a research diploid cereal model and as feedstock for the growing bioeconomy. Sorghum transformation, is technically challenging, comparatively costly and time-consuming, and limited to a few genotypes. The recalcitrance of sorghum to tissue culture and transformation is mainly attributed to genotype-dependent responses, production of phenolic compounds, short-term plant regeneration ability, and acclimatization issues (the ability of plants to survive the transfer from m vitro culture to soil) (Altpeter et al, 2016; and Maheswari et al., 2006).|OO07| The most common methods used to deliver genes into a plant genome are Agrobacterium-mediated transformation and particle bombardment (Altpeter et al., 2005b; Delporte et al., 2014b; Kooeciaftska & Wypijewski, 2001 ), both of which have proven to be extremely difficult in cereals and often genotype-dependent (Altpeter et al., 2005b; and Delporte et aL, 2014 b).|O008| With the advent of nanotechnology, various nanoparticles have been identified as vectors for foreign gene delivery into plant cells. Achieving effective gene transfection in plant cells using nanocarriers requires a mechanical approach to overcome the barrier presented by the plant cell wall. This enables the transportation of nanocarriers through the wall, using methods such as gene gun. electroporation, and ultrasound.[O009| Recently, mesoporous silica nanoparticles (MSNs) have attracted significant attention due to their unique characteristics, such as adjustable particle size, rigid structure, high pore volume, and large surface area (Li et al, 2012; Zhi et al., 2022). In addition, modifying the surface of MSNs has made them suitable carriers for controlled release of biomolecules (Choi et al., 2018; Mozafarl et al. , 2023; Pan et al., 2017), bioimaging (Lu et al., 2007), and protein and enzyme immobilization (Hao et al., 2013). MSNs have also been used for gene transfection and drug delivery into mammalian cells (Watermann & Brieger, 2017; Zhou et al., 2018; Zolghadmasab et al, 2021).|0010| There remains a long-felt, unmet need for a genotype-independent method of transforming wheat, sorghum, com, and other cereal crop varieties. In view of the foregoing, it is an object of the present disclosure to provide such a method. This and other objects and advantages, as well as inventive features, will be apparent from the description provided herein.SUMMARY[OOM | A. method of introducing a polynucleotide into a cereal seed is provided. The method comprises (i) contacting a hydrated seed of the cereal with a nanoparticle comprising the polynucleotide; and ( ii) exposing the hydrated seed of the cereal in contact with the nanoparticle comprising the polynucleotide to ultrasound to generate a transformed seed. By way of example,the cereal can. be one of wheat, sorghum, and com. The exposing step according to examples can include exposing the hydrated seed to ultrasound at an intensity of about 100 to about 800 Wand a frequency of between about 20 to about 100 kHz for a period between about 1 to about 30 minutes. In one such example, the hydrated seed is exposed to ultrasound at a frequency of about 37 kHz and the period is about 6 minutes.[0012| The met hod according to embodiments herein can further incl ude the step of preparing the hydrated seed. In one such example, preparing the hydrated seed includes the step of soaking the seed in sterile water at about 2° to about 4°C for a period ranging from about 2 days to about 14 days to generate the hydrated seed. The method according to embodiments herein can further comprise (iii) either (a) germinating the transformed seed on medium and planting the germinated seed in soil or (b) planting the transformed seed in soil.[00.13] The nanoparticle can be an inorganic nanoparticle, such as a mesoporous silica nanoparticle (MSN) or a silica nanoparticle (SN). The MSN can have a diameter of about 80 nm to about 250 nm. In certain embodiments, the MSN can have a pore size of about 2.5 nm to about 5 nm. In other embodiments, the MSN can have a pore size of at least about 15 nm. The SN can have a diameter of about 20 nm. The nanoparticle can be aminated. The nanoparticle, such as the aminated nanoparticle, can be surtace-functionalized. The nanoparticle, such as the aminated nanoparticle, can be surface -functionalized with polyethyleneimine (PEI). The method can be genotype-independent. The polynucleotide can be used for gene editing, such as CRISPR-Cas gene editing. The polynucleotide can be used for npregulating gene expression; examples of such polynucleotides include plasmid DNA (pDNA), minicircle DNA (mcDNA), synthetic mRNA, circular RNA, or self-amplifying RNA (saRNA). The polynucleotide can be used for downregulating of gene expression; examples of such polynucleotides include small interfering RNA (siRNA), antisense oligonucleotides (ASOs), short hairpin RNA (shRNA), or microRNA (miRNA).[00.14] In one particular embodiment, the nanoparticle is an MSN functionalized by PEI (MSN- PEI). In examples, the polynucleotide is a pDNA (MSN-PEI:pDNA), wherein the ratio of MSN- PEI to pDNA in aqueous mixture is about I mg MSN-PEI (e.g., 1 mg) to about 2 pg to about 10 pg pDNA (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 pg). In one such example, the ratio is 1 mg MSN- PEI to 5 tig pDNA.FIGURES[OOISJ Fig-1shows the workflow for examination of transgene expression.[001<>| Fig. 2 shows GUS-stained leaf blades of Ta generation of wheat transformed using the method.[00171 Fig- 3 shows the germination and in-pot growing o f To and Tj generati ons of sorghum plants transformed using the method. Also shown is a photograph of gel electrophoresis showing PCR-positive results for GUS and HygRresistance genes for four sorghum seedlings.[00l8| Fig. 4 shows evidence for RUBY expression in stems (left two photos) and roots (right two photos) of seedlings of sorghum plants transformed using the method.[0019| Fig. 5 shows GUS-stained roots and cross-sections of seeds of Tj (top) genera tion of corn transformed using the method.DESCRIPTION[0020| MSNs offer unique structural features such as large surface areas and tunable pore sizes, making them ideal for hosting guest molecules of various sizes, shapes, and functionalities (Torney et al., 2007), Evidence suggests that MCM-41-type MSNs can internalize drug and gene delivery in vitro to animal and plant, ceils without inducing cytotoxicity (Fang & Trewyn, 2012; Gao et al,, 2009). The first nanoparticle-based co-delivery of DN A and chemicals to Nicofiana tabacum was achieved through biolistic delivery of 100-200 nm gold-capped MSNs (Torney et al., 2007). Additionally, gold MSNs were utilized for biolistic co-delivery of DNA and proteins, such as GFPand Cre-recombinase, showcasing MSNs' capability to deliver proteins for gene editing (Martin-Ortigosa et al,, 2014),{00211 Evidence suggests that MSNs are internal ized into plant cells. Entry of MSN nanoparticles info tobacco protoplasm was demonstrated using Rhodamine B isothiocyanate- labeled APTMS-MSNs (Chang et al„ 2013). The same researchers also showed the uptake of surface-functionalized MSN particles into Arabidopsis roots (Chang et al., 2013). They demonstrated that through a simple co-culture method, MSNs can pass through the cell wall barrier and serve as a tool for transient gene expression in intact Arabidopsis roots. Other researchers were able to transfer DN A to plant leaves using MSNs (Torney et al., 2007), Mozafari et al., (2023) successfully transformed maize by MSN-mediated delivery of recombinant Ihn2f in pCAMB!A3301 (Mozafari et al., 2022, 2023). Dicot and monocot plants.however, exhibit variable degrees of direct uptake of various nanoparticle types, including MSNs (Hussain et al,, 2013), carbon nanotubes (CNTs) (Q. Liu et al., 2009), quantum dots (Koo et al., 2015), and metal / metal oxide NPs (Gonzalez-Melendi. et al., 2008; Kurepa et al. , 2010; and Larue et al,, 2012).(0022| MSN nanoparticles (1.00-250 am in diameter) have been utilized for nucleic acid delivery due to their good biocompatibility and tunable porous architecture (Kim et al, 2011 ). For instance, MSNs of approximately 100 mn with small pore sizes (2.5-5 nm) are suitable for delivering small siRNA, while MSNs of around 250 nm with large pore sizes (above 15 nm) are employed for loading large pDNA (Kim et al., 201 i ; J. Liu et al,, 2009). Nucleic add molecules are typically loaded into MSNs through weak non-covalent interactions ( Mendes et al., 2022), such as hydrogen bonding, physical adsorption, electrostatic interaction, or aromatic stacking, surface adsorption, or covalent modification.|0023| Pore size and surface functionalization play crucial roles in the loading capacity and nucleic acid release rate. MSNs with small pores offer a tunable release rate for small nucleic acids, whereas larger pores provide higher loading capacity and a faster release rate.|0024| Provided is a method of in troducing a polynucleotide into cereal seed is provided. The method comprises (1) contacting a hydrated seed of cereal with a nanoparticle comprising the polynucleotide; and (ii ) exposing the hydrated cereal seed in contact with the nanoparticle comprising the polynucleotide to ultrasound. In certain embodimems, the cereal is one of wheat, sorghum, and com. In other embodiments, the cereal is one of rice, rye, oats, millet, and barley. (0025| The method can further comprise (Hi) either (a) germinating the seed on medium and planting the germinated seed in soil or (b) planting the seed in soil. Ultrasound on the hydrated seed consistent with the present disclosure may be performed at an intensity of about 100 W to about 800 W and a frequency of between about 20 kHz to about 100 kHz for a period between about 1 minute and about 30 minutes. In various examples, the ultrasound may be performed at an intensity between about 200 W and about 700 Wtbetween about 300 W and about 600 W, between about 400 W and about 500 W, between about 500 W and about 800 W, or between about 600 W and about 800 W. In the same or other examples, frequency at which ultrasound is performed may be between about 20 kHz to about 90 kHz, about 20 kHz to about 80 kHz, about 20 kHz to about 70 kHz, about 20 kHz io about 60 kHz, about 30 kHz to about 50 kHz, or about 35 kHz to about 40 kHz. In the same or yet further examples, the duration of exposure toultrasound maybe a period between about 1 minute and about 25 minutes, a period between about 1 minute and about 20 minutes, a period between about 1 minute and about 15 minutes, a period between about 1 minute and about 10 minutes, a period between about 2 minutes and about 10 minutes, a period between about 3 minutes and about 8 minutes, or a period between about 4 minutes and about 6 minutes. As exemplified herein, in an embodiment, ultrasound may be performed on hydrated seeds at 480 W at a frequency of 37 kHz for a period of 6 minutes. [0026| Further, as exemplified herein, the seed may be hydrated prior to exposure to ultrasound and after surface sterilization, by soaking in substantially sterile water at between about 2° and 4°C for a period of time ranging from about 2 days to about 14 days, in the experimental example, the period of time ranges from about 3 days to about 7 days.{0027] The nanoparticle can be an inorganic nanoparticle, such as a mesoporous silica nanoparticle (MSN) or a silica nanoparticle (SN). The MSN can have a diameter of about SOOnm to about 250 m about 90 nm to about 250 nm, about 90 nm to about 200 nm, about 100 ran to about 250 nm, or about 100 nm to about 200 nm. In certain embodiments, the MSN can have a pore size of about 2.5 nm to about 5 nm. In other embodiments, the MS N can have a pore size of a t least about 15 nm. The SN can have a diameter of about 20 nm. The nanoparticle can be aminated. The nanoparticle, such as the aminated nanoparticle, can be surface-functionalized. The nanoparticle, such as the aminated nanoparticle, can be surface-functionalized with polyethyleneimine ( PEI). Examples of other cationic polymers include polyamidoamine, polylysine, poly(allylanime), and polyfdiallydimethylammonium chloride). Other examples can be found in Polymer Handbook, 4il!cd., Brandrup et al,, eds, John Wiley & Sons (1999). The method can be genotype-independent. The polynucleotide can be used for transgene insertion or gene editing, such as CRISPR (clustered regularly interspaced short palindromic repeats)-Cas gene editing (see, e.g., USPN 11,518,999, col. 4, 1. 63, through col. 9, 1. 2, which is hereby specifically incorporated by reference for its teachings regarding same). The polynucleotide can be used for upregulatmg of gene ex pression; examples of such polynucleotides include plasmid DNA (pDNA), minicircle DNA (mcDNA), synthetic mRNA, circular RNA, or self-amplifying RNA (saRNA). 'Hie polynucleotide can be used for downregulating of gene expression; examples of such polynucleotides include small interfering RNA (siRNA), antisense oligonucleotides (ASOs), short hairpin RNA (shRNA), or microRN A (mlRN A). The polynucleotide can be multiple plasmid DNAs or linearized DNAs, each carrying a purpose-driven gene or regulatory element, with the expectation that all polynucleotides are integrated into the recipient genome. The plasmid DMA can be any one of those meant for regular gene expression in biolistic and other non- Agrobacterium plant transformation technologies or any of those based on T-DNA binary vectors used in Agrobacterium systems.[0028| In certain embodiments, the functionalized nanoparticle is MSN-PEI. In examples, the ratio of MSN-PEI to polynucleotide in aqueous mixture may be about 1 mg MSN-PEI to about 2-10 pg polynucleotide, about 1 mg MSN-PEI to about 3-8 pg polynucleotide, about 1 mg MSN- PEI to about 4-7 pg polynucleotide, or about 1 mg MSN-PEI to about 5-6 pg polynucleotide. Further, as exemplified herein, the polynucleotide can be pDNA resulting in MSN-PEI rpDN A. In examples, the ratio of MSN-PEI to polynucleotide in aqueous mixture may be about I mg MSN-PEI to about 2-10 pg pDNA, about 1 mg MSN-PEI to about 3-8 pg pDNA, about 1 mg MSN-PEI to about 4-7 pg pDNA, or about 1 rag MSN-PEI to about 5-6 pg pDNA. In the exemplified embodiment, the ratio is 1 mg MSN-PEI to 5 pg pDN A.EXPERIMENTAL DESCRIPT IONNanoparticle Preparation

[0029] Two types of nanoparticles, with different sizes based on TEM data provided by the manufacturing company nanoCoraposix, Fortis Life Sciences (San Diego, CA), were used to deliver plasmid DN A (pDN A) into wheat with the goal of achieving stable transformation. The first material used was aminated silica nanospheres (5W) (SKtl: SIAN20-25M), with TEM particle sizes of 22.4+2,6 nm (with coefficient of variation of 11,4 % %), a surface area of 1 18,6 tn2 / g, and Zeta potential of > 68.8 mV after amination. The second material was aminated mesoporous silica nanospheres (MSV) (Si l AD 100) from the same vendor with TEM reported particle size of 91 nm (coefficient of variation of 15.8%), a surface area of 667 m2 / g, and pore diameter and volume of 3. 18 nm and 0.599 cmVg, respectively. These MSNs were surface functionalized by poly( ethylene imine) ’amine', hence referred to as PEl-MSNs.|0030| To prepare nanoparticles for transformation, 200 uL of 70% ethanol were added to the PEI-SNs and PEl-MSNs, followed by incubation at 4 “C for 16 hours. The nanopanicles were then centrifuged at 5000* g for 10 minutes and washed three times with DEPC-treated water (10 minutes at 5000* g) to remove excess ethanol. Subsequently, 500 pL of Dulbecco's phosphate- buffered saline (DPBS) buffer were added to the washed nanoparticles.100311 The experimental foreign DNAused for transformation in this study was a plasmid DMA (pDNA) pCAMBl Al 305.1 construct. This pDNA harbors a GUS-plus™ gene and a hygromycin resistance HygR gene pCAMBIAI 305.1 is a binary vector used for transfonnation mediated by Agrobacterium. However, nanomaterial-based gene transfer does not need the components of a binary vector. Large DNA segments, e.g., comprising slacked genes, native genes, or transgenes, can be inserted into the genome of a plant without the need for Agrobacterium.[0032| Among the two nanoparticle types used, initial results were obtained with the MSNs and not SNs. A range of pDNA quantities (from 5 gg / mL to 20 pg / mL) was tested in 500 pl. DPBS buffer added to the prepared nanoparticles in DPBS, resulting in 1 mL of PEI-MSNs:pDNA mix. Gel shifting assay was utilized to demonstrate the conjugation of nano-pDNA elements. As additional proof of transformation, a second vector harboring another reporter gene called RUBY, which converts the amino acid tyrosine into a vivid red color betalain (He et al.. 2020), was examined.Wheat Transformation|0033| Wheat (Trificum aeMivum), namely Apogee, Sonora-64, and Yecora rojo, were used. To prepare seeds for transformation, a gentle physical treatment was performed to enhance seed membrane and cell wall permeability. Initially, seeds were surface-sterilized rising a halfstrength commercial bleach solution multiple times, followed by extensive rinsing to remove excess bleach. Then, a prolonged wet-cold pre-treaiment involved soaking the seeds in sterile water at 40C for a period ranging from 3 to 7 days.[00341 The actual transformation procedure involved using a 2 ml, tube and soaking the pretreated seeds in the 1 mL PEI-MSNs:pDNA mix prepared earlier and gently mixing at room temperature for 2 hours. After incubation, the mixture underwent ultrasound treatment using a water bath ultrasound with an intensity of 100% power (480 W) and frequency of 37 kHz for 6 minutes in an Ultrasonic Bath mode! (FB-11201 , Fisherbrand™). All steps were conducted under plant genetic laboratory1conditions (e.g., I.SPS 213). The seeds were then germinated on plates and transplanted to soil in a greenhouse. Leaf samples from seedlings were utilized for PCR tests and GUS staining.{0035} Segregation analysis was conducted to test whether the transgene insertions are transmitted to subsequent generations. Progeny from a Ti "Apogee5plant was used for this analysis. Twenty-three TJ seedlings were assayed histochemically for GUS expression six daysafter germination. Of these, 21 seedlings exhibited blue staining, while two seedlings showed no detectable staining, corresponding to approximately 91% GUS-positive individuals.[0036| The workflow for examination of transgene is shown in Fig, 1 for genotype Apogee, as well as sorghum (Sargbum bicotor) and corn (Zea mays) herein. In 102, multiple TO plants were grown, and seeds were harvested. In 104, one ripened seed per each TO plant was planted individually. Upon positive PCR for hygromycin resistance and GUS gene, in 106, seeds from each Ti were pooled, and T2 generations were planted in bulk of 6-8 seedlings. Finally, in 108, the 1'2 generations showed PCR-positive for hygromycin resistance and GUS genes. They also showed GUS staining phenotype. Fig. 2 shows GUS-stained leaf blades of the T2 generation of wheat transformed using the method herein described.(0037] This technology has successfully led to the expression of transgenes, specifically the GUS reporter gene p-glucuronidase, in wheat.Sorghum 'rransformation[0038| .W ghum bicolar was used. Seeds of experimental lines ‘P 1253B’ and ‘P9401’ were generously provided by Dr. Gebisa Ejeta, Purdue’s sorghum genetics and breeding program. To prepare seeds for transformation, a gentle physical treatment was performed to enhance seed membrane and cel l wall penneability. Initially, seeds were surface-sterilized using a halfstrength commercial bleach solution multiple times, followed by extensive rinsing to remove excess bleach. Then, a prolonged wet-cold pre-treaiment involved soaking the seeds in sterile water at 40C for a period ranging from 3 to 7 days.

[0039] The actual transformation procedure involved using a 2 ml, tube and soaking the pretreated seeds in the 1 mL PEI-MSNs:pDNA mix prepared earlier and gently mixing at room temperature for 2 hours. After incubation, the mixture underwent ultrasound treatment using a water bath ultrasound with an intensity of 100% power ( -480 W) and frequency of 37 kHz for 6 minutes in an Ultrasonic Bath model (FB- 11201 , Fisherbrand™). All steps were conducted under plant genetic laboratory1conditions (e.g., 1..SPS 213). The seeds were then germinated on plates and transplanted to soil in a greenhouse. Leaf samples from seedlings were utilized for PCR tests and GUS staining.[0040J The workflow for examination of transgene for genotype P9401 is as follows. .Multiple TO plants were grown, and seeds were harvested. One ripened seed per each TO plant was planted individually. Upon positive PCR for hygromycin resistance and GUS gene, seeds fromeach T1 were pooled, and T2 generations were planted in bulks of 6-8 seedlings. The T2 generations showed PCRpositive for hygromycin resistance and GUS genes. They also showed GUS staining phenotype . Fig. 3 shows the germination and in-pot growing of To and Ti generations of sorghum plants transformed using the method. Also shown is a photograph of gel electrophoresis showing PCR-positive results for GUS and HygRresistance genes for four sorghum seedlings.

[0041] This technology has successfully led to the expression of transgenes, specifically the GUS reporter gene ^-glucuronidase, in sorghum. In particular, Fig. 4 presents evidence for RUBY expression in stems (left two photos) and roots (right two photos) of seedlings of sorghum plants transformed using the method.Com Transformation|0042| Corn (Zea mays)' seeds of single hybrid cross were used. To prepare seeds for transformation, a gentle physical treatment was performed to enhance seed membrane and cell wall permeability. Initially, seeds were surface-sterilized using a half-strength commercial bleach solution multiple times, followed by extensive rinsing to remove excess bleach. Then, a prolonged wet-cold pre-treatment involved soaking the seeds in sterile water at 4*C for a period ranging from 3 to 7 days.[0043| The actual transformation procedure involved using a 15 mL falcon tube and soaking the pre-treated seeds in the 1 ml. PEI-MSNs.pDNA mix prepared earlier and gently mixing at room temperature for 2 hours. After incubation, the mixture underwent ultrasound treatment using a water bath ultrasound with an intensity of 100% power (-480 W) and frequency of 37 kHz for 6 minutes in an Ultrasonic Bath model (FB- l 1201, Fislierbrand™). All steps were conducted under plant genetic laboratory conditions (e.g., LSPS 213), The seeds were then germinated on plates and transplanted to soil in a greenhouse. Leaf samples from seedlings were utilized for PCR tests and GUS staining.

[0044] The workflow for examination of transgene is as fol lows. Multiple TO plants are grown, and seeds are harvested. One ripened seed per each TO plant is planted individually. Upon positive PGR for hygromycin resistance and GUS gene, seeds from each T1 are pooled, and T2 generations are planted in bulks of 6-8 seedlings. The 12 generations are subjected to PCR for examination of hygromycin resistance and GUS gene expression. The T2 generation are alsoexamined for GUS staining phenotype. Fig. 5 shows GUS-stained roots and cross-sections of seeds of Tf (top) generation of coni transformed using the method.

[0045] This technology can lead to the expression of transgenes, specifically the GUS reporter gene p-glucuronidase, in corn.ReferencesAltpeter, F., Baisakh, N.. Beachy, R., Bock, R,, Capell, T., Christou, P., Daniell, H., Datta, K., Datta, S., Dix, I’. J., Fauquet, C., Huang, N., Kohli, A., Mooibroek, H., Nicholson, L„ Nguyen, T, T., Nugent, G., Raemakers, K., Romano, A., ... Visser, R. (2005a). Partide bombardment and the genetic enhancement of crops: Myths and realities. In Molecular Breeding (Vol. 1.5, Issue 3, pp. 305 -327). https: / / doi.org / 10. 1007 / s 11032-004-8001 -yAltpeter, F., Baisakh, N., Beachy, R., Bock, R., Capell, T., Christou, R, Daniell, H., Datta, K., Datta, Dix, R J., Fauquet, C., Huang, N,, Kohli, A,, Mooibroek, H., Nicholson, L., Nguyen, T. T,, Nugent, G., Raemakers, K., Romano, A., ... Visser, R. (2005b). Particle bombardment and the genetic enhancement of crops: myths and realities. 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J., Laidlaw And, H. K. E,, & Godwin, I. D. (2004). PARTICLE INFLOW GUN-MEDIATED TRANSFORMATION OF SORGHUM RICOLOR.Zhi, H., Zhou, S., Pan, W., Sbang, Yi, Zeng; Z., & Zhang, H. (2022). The Promising Nanovectors for Gene Delivery in Plant Genome Engineering. In International Journal of Molecular Sciences (Vol. 23, Issue 15), MDPI. httj3s: / / doi.org / H).3390 / ijms23 l58501Zhou, Y.» Quan, G., Wu, Q., Zhang, X., Niu, B., Wu, B., Huang, Y, Pan, X., & Wu, C. (2018).Mesoporous silica nanoparticles for drug and gene delivery. Aeta Pharmaceutiea Sinlca il, 8(2), 165-177. MmsVAy .oro / i 6. i 0 ; 6 / i.ansb.2018.01 067Zolghadmasab, M., Mousavi, A., Farmany, A., & Arpanaei, A. (2021 ). Ultrasound-mediated gene delivery into suspended plant cells using polyethyleueimine-coated mesoporous silica nanoparticles.Ultrasonics Sonochemistiy, 73, 105507, https : / / doi ,org / 10.1016(j .ul tsonch.2021.105507All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those in the art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and indi vidually indicated to be incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.The invention illustratively described herein may be suitably practiced in the absence of any elements) or Iirnitat.ion(s), which is / are not specifically disclosed herein. Thus, for example, each instance herein of any of the terms "comprising, ’’ ’’consisting essentially of,’* and ’’consisting of may be replaced with either of the other two terms. Likewise, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" include one or more methods and / or steps of the type, which are described herein and / or which wil l become apparent to those ordinarily skilled in the art upon reading the disclosure. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art. The following termsand phrases shall have the meaning indicated.The term "about," when referring to a number or a numerical value or range (including, for example, whole numbers, fractions, and percentages ), means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) and thus the numerical value or range can vary between 1% and 15% of the stated number or numerical range (e.g„ + / - 5 % to 15% of the recited val ue, such as within 10%. within 5%, or within 1% of a stated value or stated limit of a range) provided that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). The term "substantially" can allow for a degree of variability in a value or range, for example, within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or of a stated limit of a range.In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section beading may occur within or outside of that particular section.

Claims

WHAT IS CLAIMED IS:

1. A method of in troducing a polynucleotide into a cereal, which method comprises:( i ) contacting a hydrated seed of the cereal with a nanoparticle compri sing the polynucleotide; and(ii) exposing the hydrated seed in contact with the nanoparticle comprising the polynucleotide to ultrasound to generate a transformed seed.

2. The method of claim 1 , wherein the cereal is one of wheat, sorghum , and corn .

3. The method of claim 2, wherein the nanoparticle is a mesoporous silica nanoparticle (MSN), wherein the MSN surface is functionalized by polyethylene imine) (PEI) (MSN-PEI),4. The method of claim 3, wherein the polynucleotide is a pDNA(MSN-PEI:pDNA).

5. The method of claim 4, wherein the ratio of MSN-PEI :pDNA is 1 mg MSN-PEI to about 2 ug to about 10 pg pDNA.

6. The method of claim 5, wherein the ratio of MSN-PEI:pDNA is 1 mg MSN -PEI to 5 pg p'DNA.

7. The method of any of claims 1-6, wherein the method further comprises preparing the hydrated seed, wherein the preparing comprises soaking a seed in sterile water at about 2° to about 4'-'C for a period of time ranging from about 2 days to about 14 days to generate the hydrated seed.

8. The method of any of clai ms 1-6, wherein the exposing comprises exposing the hydrated seed to ultrasound at an intensity of about 100 W to about 800 W and a frequency of about 20 kHz to about 100 kHz for a period of time of about 1 minute to 30 minutes.

9. The method of claim 8, wherein the intensity is about 480 W at a frequency of about 37 kHz for a period of about 6 minutes.

10. The method of claim 1 , which further comprises:( i i i) either (a) germinating the transformed seed on medium and planting the germinated seed in soil or (b) planting the seed in soil.11 . lire method of claim 1. wherein the nanopanicl e is an inorganic nanoparticle.

12. The method of claim 11 , wherein the inorganic nanoparticle is a mesoporous silica nanoparticle (MSN).

13. The method of any one of claims 3-6, or 1:2, wherein the MSN has a diameter of about 80 ran to about 250 ran.

14. The method of claim 13, wherein the MSN has a pore size of about 2.5 ran to about 5 mn.

15. The method of claim 13, wherein the MSN has a pore size of at least about 15 ran.

16. The method of claim 12, wherein the inorganic nanopanicle is a silica nanoparticle (SN).

17. The method of claim 16, wherein the SN has a diameter of about 20 nm.

18. The method of claim I , wherein the nanoparticle is aminated.

19. The method of claim 1 . wherein the nanoparticle is surface-functionalized.20 The method of claim 19, wherein the nanoparticle is surface-functionalized with po 1 yethylenei m ine.21 . The method of any one of claims I -6, which is genotype-independent.

22. The method of any one of claims 1-6 or 21 , in which the polynucleotide is used for gene editing and / or transgene insertion.

23. The method of claim 22, wherein the gene editing is CRISPR-Cas.

24. The method of any one of claims 1-6 or 21 . in which the polynucleotide is used for upregulating of gene expression.

25. The method of claim 24, wherein the polynucleotide is plasmid DNA (pDNA), minicircle DNA(mcDNA), synthetic mRNA, circular RNA, or self-amplifying RNA (saRNA).

26. The method of any one of claims 1-6 or 21, wherein the polynucleotide is used for downregulating of gene expression.

26. The method of claim 26, wherein the polynucleotide is small interfering RNA (siRNA), antisense oligonucleotides (ASOs), short hairpin RNA (shRNA), or microRNA (mi RNA).

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