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Table 1 Summary of various leaf infiltration techniques and their applications

From: Leaf infiltration in plant science: old method, new possibilities

Field of plant science

Application

The purpose of the leaves infiltration

Infiltrated substances

Method used for leaf infiltration/plant material

Selected infiltrated species; additional informationa

Referencesb

 

Studies using output infiltration methods

Molecular plant physiology, apoplast studies, OMICS

Secretome sampling from plants growing in the artificial conditions and (or) in the natural environment

Leaf infiltration is the first step in the infiltration-centrifugation procedure. The method allows the recovery of AWF from the intercellular spaces of plant leaves

Solutions with diverse composition and pH, precisely selected for the experimental needs, e. g., pure water or multi-component buffers containing additional substances such as protease inhibitors in the case of proteomic studies. Markers, such as malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI) or glucose-6-phosphate dehydrogenase (G6PDH), are often added to the infiltration buffer to enable the quality assessment of the isolated AWF

Vacuum infiltration using 50–60 ml syringe or vacuum pump/whole leaves infiltration directly on the mother plant, harvested whole leaves and leaf fragments

Vitis vinifera cv. ‘Trincadeira’, V. vinifera cv. ‘Regent’

[37]

Arabidopsis thaliana

[44]

Prunus persica cv. Wanxifei

[53]

Zea mays

[45]

hybrid poplar clone ‘546’: Populus deltoides cv. 55/56 × P. deltoides cv. Imperial

[52]

Faseolus vulgaris, Solanum lycopersicum, A. thaliana

[2]

Oriza sativa, Triticum aestivum, Phaseolus vulgaris, Spinacia oleracea

[48]

Vicia faba, S. oleracea, Beta vulgaris, Z. mays

[6]

Molecular plant physiology, microbiology

Isolation of microorganisms inhabiting the leaf apoplastic spaces or isolation of AWF as a component of media for the cultivation of endophytic microbes

Lactuca sativa L. var. crispa cv. Salinas—Salmonella enterica Serovar Typhimurium

[47]

Z. mays—Pantoea stewartii subsp stewartii

[45]

A. thaliana—Pseudomonas syringae pv. tomato DC3000, S. lycopersicum var. cerasiforme cv. ‘Tenten’

[11]

Plant biotechnology, molecular pharming

Study of recombinant proteins secreted into the apoplast of transgenic plants

Composition and pH of the infiltration buffer selected for the most effective recovery of recombinant proteins; common components: Tris-NaCl (20–100 mM; pH 5.5–7.7), MgCl2 (10 mM), EDTA (2 mM), NaCl (100 mM), NaOAc (20 mM), sodium metabisulphite (4 mM)

Nicotiana tabacum—recombinant human deoxyribonuclease I (rhDNaseI)

[54]

N. benthamiana—griffithsin (GRFT)

[92]

N. benthamiana—recombinant IgA with defined N- and O-glycans

[56]

N. tabacum—thermostable xylanase from Clostridium thermocellum

[55]

 

Studies using input infiltration methods

Plant—microorganism—environment relationships, phytopathology

Plant-phytopathogen interactions study. Etiology of plant diseases. Investigation of plant defense mechanisms

Leaf infiltration with suspension of phytopathogen cells as method for controlled induction of plant infection

Suspension of phytopathogens or non-pathogen endophyte cells in water or properly selected infiltration medium, most often composed of MES (10 mM) and magnesium salt (MgCl2 or MgSO4, 10mM).

Spontaneous infiltration, forced infiltration/whole leaves on the mother plant, whole harvested leaves, leaf fragments

Citrus × limonia—Xanthomonas citri subsp, citri

[66]

A. thaliana—P. syringae pv. tomato DC3000

[4]

A. thaliana, Solanum lycopersicum var. cerasiforme cv. ‘Tenten,’—P. syringae pv. tomato DC3000

[11]

A. thaliana—P. syringae pv. maculicola ES4326

[5]

Plant—microorganism—environment relationships, microbial ecology

Leaf microbiome studies, endophyte-plant-phytopathogen tripartite interactions and their importance for the overall health of a plant

Introducing suspension of various microorganisms, e.g., endophytic bacteria into the intercellular leaf spaces

A. thaliana - Blumeria graminis f. sp. Hordei

[44]

A. thaliana, S. lycopersicum var. cerasiforme cv. ‘Tenten—Bacillus cereus (GU982920.1), Variovorax paradoxus (JN990697.1), Rhodococcus kyotonensis (AB920569.1), R. corynebacteriodies (AY438619.1)

[11]

Applied microbiology, agricultural microbiology

Design and testing of plant protection products containing microorganism with an antagonistic effect towards phytopathogens

Citrus × limonia—Pseudomonas entomophila, Xanthomonas citri subsp, citri; Bacillus amyloliquefaciens LE109

[66]

S. lycopersicum var. cerasiforme cv. ‘Tenten—P. syringae pv. tomato DC3000, B. cereus (GU982920.1)

[11]

Applied microbiology, food technology

Study of the activity of human pathogens in the contamined tissues of edible plants especially leafy vegetables

The cotrolled introduction of human pathogens into the tissues of leafy vegetables

L. sativa L. var crispa cv. Salinas—Salmonella enterica Serovar Typhimurium

[47]

L. sativa L.—Escherichia coli O157: H7 GFPlux

[20]

Plant biotechnology, in vitro cultures

Production of stable transformed plant lines and lines with modified genome

The spontaneous infiltration of leaf explantates with Agrobacterium is the first step in the generation of transgenic plants using agrotransformation in vitro. The use of forced infiltration of explantates can additionally increase the effectiveness of the transformation

For spontaneous in vitro infiltration, a suspension of Agrobacterium in a culture medium (e.g., YEB) is commonly used (with the addition of acetosyringone (100–200 µM) optionally). In the case of forced infiltration, the Agrobacterium cells are usually re-suspended in a medium of the same composition as that used for the transient transformation of plants by agroinfiltration

Spontaneous infiltraction, forced infiltration/leaf fragments, cotyledons

A. thaliana, Fragaria vesca cv. ‘YW5AF7’, N. benthamiana—GFP

[9]

Malus domestica cv. ‘Gala’; Pyrus communis cv. ‘Conference’—GUS

[71]

Taraxacum officinale - GUS

[72]

N. tabacum—CRISPR-Cas9-mediated knockout of NtFAD2-2

[36]

Plant biotechnology, transient plant transformation using agroinfiltration

Generation of transient transformed leaf tissues or genome edition for functional characterisation of genes in planta

Infiltration/co-infiltration of intercellular leaf spaces with a suspension containing Agrobacterium carrying the target genes

Agrobacterium suspension in infiltration medium containing MES (10 mM, pH 5.5–5.6), MgSO4 or MgCl2 (10 mM), and possibly additional substances to increase the transformation efficiency, e.g., acetosyringone (100–150 µM); 5-azacydine AzaC (20 µM), ascorbate acid (0.56 mM), DTT (0.5 mM); surfactants, e.g., Tween 20 (0.015–0.03%), Silwet L-77 (0.01%), Triton™ X (0.001%)

Forced infiltration/whole plants, intact leaves direct on mother plants, whole harvested leaves, leaf fragments

Populus clones—GFP, LUC, GUS, CBL1, MTP1, C4H, GT47C, MYB221, and PrxQ, The coding regions of three key activators of secondary cell wall biosynthesis, Populus davidiana × P. bolleana

[89]

Canabis sativa—GUS; The phytoene desaturase gene was silenced with a transient hairpin RNA expression, resulting in an albino phenotype in the leaves

[35]

Sorghum bicolor, GFP

[87]

N. benthamiana—sucrose transporters StSUT1, StSUT2, StSUT4 and their interaction partner SNARE/VAMP

[31]

N. benthamiana—marker genes under the control of the optogenetic system PULSE

[76]

Luffa cylindrica—GUS

[28]

N. benthamiana—GFP, GUS, mouse granulocyte-macrophage colony-stimulating factor, and human fibroblast growth factor 1

[8]

Medicago truncatula cv. R108, N. benthamiana—MYB transcription factors: MtLAP1, a MYB transcription factor involved in the regulation of the anthocyanin pathway, various TF flowering time regulators, AcMYB10 or 35S:AtLEC2

[75]

N. benthamiana—GUS

[32]

N. benthamiana—cooexpression of GFP-Cas9 and sgRNA with the guide sequence within the PDS gene under U6 promoter

[77]

S. tuberosum cv. Russet Burbank, S. tuberosum cv. Shepody—TALENs targeted to endogenous starch branching enzyme and an acid invertase

[78]

Dendrobium catenatum Lindl.—GFP, GUS

[36]

Glycine max, N. benthamiana, GUS

[84]

Generation of transiently transformed leaf tissues or genome edition to create plant bioreactors capable of producing recombinant proteins with potential utility, e.g., in medicine, industry, agriculture

N. benthamiana ΔXT/FT—ACE2-Fc fusion protein consisting of the Angiotensin-Converting Enzyme 2 (ACE2)—the primary host cell receptor for SARS-CoV-2 binding and the fragment crystallizable (Fc) of human IgG

[96]

N. benthamiana—capsid protein of hepatitis B virus; HBc

[80]

N. benthamiana—SARS-CoV-2 receptor binding domain (RBD), spike specific monoclonal antibody CR3022

[97]

N.benthamiana—mAbs B38 and H4 neutralizing SARS-CoV-2

[98]

N. benthamiana—ΔXT/FT—cooexpression of component proteins of Human IgA Isotypes

[90, 101]

N. benthamiana—hepatitis B core antigen (HBcAg)

[81]

N. tabacum, L. cylindrica—codon optimised human deoxiribonuclease I; (Dnase I)

[54]

N. benthamiana—GRFT, SP-D, CV-N, hMBL, galectin-9

[92]

N. benthamiana—the recombinant sIgA1 was either expressed alone or co-infiltrated with the vectors carrying genes encoding the proteins for N-glycan modification or mucin-type O-glycosylation

[56]

Plant biotechnology, nanobiotechnology

Generation of nanobionic plants to improve their natural functions or give new properties

Introducing a suspension of nanoparticles to the target plants.

The specially prepared nanomolecules resuspended in infiltration medium. Common components: MES (10 mM) or TES (10 mM), MgCl2 (10 mM) and surfactants, e.g., Silwet L-77 (0.05%)

Spontanaceous infiltration, forced infiltration/whole leaves on the mother plants

S. oleracea, O. sativa, Pteris cretica—SWINT-based optical nanosensors

[106]

Gossypium hirsutum L. var. Xinluzao 74, XLZ 74 - poly acrylic acid coated nanoceria; PNC

[27]

S. oleracea, Blitum capitatum, L. sativa, Rumex acetosa, Eruca sativa, A. thaliana—SWNT-based optical nanosensors

[105]

A. thaliana—cerium oxide nanoparticles; nanoceria

[107]

A. thaliana—TGA-QD

[104]

  1. aFor microbiology references, the studied microorganisms names are listed; for references describing transgenic plant modifications, the studied genes or recombinant proteins are listed; for nanobiotechnology references the used nanomolecules are listed
  2. bWith particular emphasis on the most recent publications
  3. AWF apoplast washing fluid; AzaC 5-azacytydine; C4H cinnamate-4-hydroxylase; CBL1 calcineurin B-like calcium sensor protein 1; CV-N cyanovirin-N; GFP green fluorescent protein; GT47C glycosyl-transferase family 47; Nt FAD2-2 microsomal Δ12 oleate desaturase (1-acyl-2-oleoyl-snglycero-3-phosphocholine Δ12 desaturase); GRFT griffithsin; GUS beta-glucuronidase; Hmbl human mannose-binding lectin; IgA immunoglobulin A; IgG immunoglobulin G; LUC firefly luciferase; MES 2-(N-morpholino)ethanesulphonic acid; MtLAP1 M. truncatula legume anthocyanin producition 1; MTP1 metal-tolerance protein 1; PDS phytoene desaturase; Prxq chloroplastic peroxiredoxin Q; SP-D surfactant protein D; SWINT single-walled carbon nanotubes; TES 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid; TGA-QD thioglycolic acid quantum dots