Table 4.

Application of external RNAs for the suppression of plant transgenes or endogenous genes.

Target	RNA Treatment	RNA Amount	RNA Application	Plant Host	Effect Assessment	Effect Maintenance	Reference
Plant Transgenes
YFP transgene	In vitro synthesized short dsRNA (21 bp) in a complex with a carrier peptide	100 µL of the RNA-peptide complex (20 pmol siRNA)	Infiltration of the complex into intact plant leaf cells using a syringe without a needle	Arabidopsis, poplar	Suppression of YFP protein level and fluorescence	At least for 24–36 hpt	Numata et al. (2014) [24]
GFP transgene	In vitro synthesized siRNAs	100 µL of aqueous siRNA solutions (10 µM)	High-pressure spraying (using a conventional compressor and an air brush pistol) at the abaxial surface of leaves	Tobacco	Local and systemic GFP fluorescence suppression (detected 2–20 dpt)	Up to 20 dpt	Dalakouras et al. (2016) [25]
GUS transgene	Total RNA from dsRNA-expressing bacteria (~504 bp)	100 µg of dsRNA with or without LDH	Sprayed with an atomizer	Arabidopsis	Reduction in GUS activity	Assessed 7 dpt	Mitter et al. (2017) [10]
EGFP and NPTII transgenes	In vitro synthesized dsRNAs (EGFP 720 bp; NPTII 599 bp)	0.35 µg/µL (100 µL per 4-week-old plant)	Spreading with sterile individual soft brushes	Arabidopsis	Suppression of EGFP and NPTII mRNA levels; suppression of EGFP protein level and fluorescence; induction of EGFP and NPTII DNA methylation	At least for 7–14 dpt	Dubrovina et al. (2019) [26]
Plant Endogenous Genes
EPSPS gene	In vitro synthesized short dsRNAs (24 bp); long dsRNAs (200–250 bp)	10 µL of dsRNA on each of four leaves per plant (0.024–0.8 nM)	Leaves pre-treatment by carborundum solution or surfactant solution	Palmer Amaranth (glyphosate-tolerant)	Suppressed EPSPS transcript and protein levels; improved glyphosate efficacy	at least for 48–72 hpt	Sammons et al. (2011) [68]
CHS gene	In vitro synthesized short dsRNA (21 bp) in a complex with a carrier peptide	100 µL of protein carrier in a complex with the siRNA (6 pmol)	Infiltration of the complex into intact plant leaf cells using a syringe without a needle	Arabidopsis	Local loss of anthocyanin pigmentation	Assessed 2 dpt	Numata et al. (2014) [24]
STM and WER genes	A mixture of cationic fluorescent nanoparticles G2 and in vitro synthesized dsRNA (STM 450 bp; WER 550 bp)	1 µg of dsRNA mixed with 3 µg of gene carrier G2 per root of Arabidopsis once every 24 h (3 days of treatment)	By pipette	Arabidopsis	Suppressed transcripts of STM and WER; retarded growth and reduced meristem size; fluorescence observed throughout the root system (24 hpt)	at least for 5–7 dpt	Jiang et al. (2014) [27]
MYB1 gene	Crude bacterial extract containing DhMYB1 dsRNA (430 bp)	50 μL of crude bacterial extract (2 μg/μL, at 5 day intervals)	Mechanical inoculation (gently rubbing onto a flower bud using a latex-gloved finger)	hybrid orchid	Suppressed expression of DhMYB1; changed phenotype of floral cells (22, 25, and 29 dpt)	at least for 29 dpt	Lau et al. (2015) [28]
Mob1A, WRKY23, and Actin genes	In vitro synthesized dsRNA (Mob1A 554 bp; WRKY23 562 bp)	Arabidopsis and rice seeds or seedlings soaked in 0.2 or 1 mL dsRNA (1.0 mg/mL)	Root soaking	Arabidopsis, rice	Absorption of the dsRNA by plant roots; suppressed target genes; suppression of the root growth and seed germination; plants could not bolt or flower	at least up to 5–7 dpt	Li et al. (2015) [20]

YFP—yellow fluorescent protein; GFP—green fluorescent protein; dpt—days post treatment; hpt—hours post treatment; GUS—β-glucuronidase; EGFP—enhanced green fluorescent protein; NPTII—neomycin phosphotransferase II; EPSPS—5-enolpyruvylshikimate-3-phosphate synthase; CHS—chalcone synthase; STM—class I knotted-like homeodomain protein SHOOT MERISTEMLESS; WER—a R2R3-type MyB-related transcription factor WEREWOLF.