Sterilization in Plant Tissue Culture Using Nanoparticles: What Works, Why it Works, and How to Use It.

Keeping cultures contamination-free is the hardest part of plant tissue culture. Traditionally we use sodium hypochlorite (NaOCl), ethanol, or even toxic mercuric chloride (HgCl₂). In the last decade, nanoparticles (NPs) especially silver nanoparticles (AgNPs) have emerged as powerful alternatives for surface sterilization of explants and keeping the culture medium clean. They often kill microbes at lower doses, sometimes improve growth, and avoid mercury altogether. (RSC Publishing)

How nanoparticles help against contamination

• Broad antimicrobial action: AgNPs and some metal-oxide NPs (ZnO, TiO₂, CuO) disrupt cell walls, damage membranes and DNA, and release metal ions that kill fungi and bacteria even endophytes that survive classic bleach/mercury dips. (RSC Publishing)
• Two routes of use:

1.Quick dips of explants in an NP solution before culture (surface disinfection).

2.Low doses in the culture medium (or as a thin “bilayer” on top) to suppress microbes that escape disinfection. (SpringerLink)

• Bonus growth effects (sometimes): AgNPs can reduce ethylene inside vessels and improve shoot multiplication and leaf area; results depend on species and dose. (SpringerLink, e-services.nafosted.gov.vn)

What the research shows

• AgNPs vs HgCl₂ (classic disinfectant): Explants treated with 200 mg/L AgNPs for ~20 min often had better disinfection and better shoot induction than 1,000 mg/L HgCl₂ for 5 min (e.g., Limonium sinuatum, strawberry). (SpringerLink, e-services.nafosted.gov.vn)
• Add to medium at low dose: In Psidium friedrichsthalianum, a 5 mg/L AgNP bilayer on the medium cut contamination (~40–50%) and increased leaf area (~5.6×) and multiplication rate (~1.8×). (SpringerLink)
• Roses and grapes: 200 mg/L AgNPs for 20 min reduced bacterial contamination of Rosa hybrida explants; adding ~100 mg/L to medium further reduced contamination and phenolic exudation. Similar effects reported in grapevine. (RSC Publishing, ResearchGate)
• Banana endophytes problem: When standard bleach + alcohol + HgCl₂ failed, 100 mg/L Zn or ZnO NPs in medium produced contamination-free banana shoot tips. (RSC Publishing)
• Callus cultures: 20–40 mg/L AgNPs in medium removed fungi/bacteria from culture media in several systems; TiO₂ NPs (~60 μg/mL) also eliminated bacterial contamination after a few subcultures in barley callus. (PMC, RSC Publishing)
• Scoparia dulcis endophytes: Casein-stabilized Ag, Au, and CuO NPs (≈4 mg/L) reduced endophytic contamination and influenced callus/shoot regeneration (CuO often strongest). (Semantic Scholar)

Practical “how-to”

Always run small pilot tests by species and explant type. Start low, observe phytotoxicity, and adjust.

A) Surface sterilization (pre-culture dip)

1.     Do a gentle pre-wash (detergent + water), then 70% ethanol 30–60 s.

2.   Dip explants in AgNP solution (trial: 100–200 mg/L) for 10–20 min, rinse with sterile water (2–3×), and inoculate. Species like rose and strawberry responded well at 200 mg/L for 20 min. (ResearchGate, e-services.nafosted.gov.vn)

B) In-medium protection

• For persistent contamination/endophytes, supplement the medium:
• AgNPs: try 1–10 mg/L (some systems used ~5 mg/L bilayer on semisolid medium). (SpringerLink)
• Zn/ZnO NPs: ~100 mg/L has rescued banana shoots. (RSC Publishing)
• TiO₂ NPs: tens of μg/mL reported in callus systems. (RSC Publishing)

C) When to stop or step down

• If you see chlorosis, stunting, or reduced regeneration, reduce NP dose or remove NPs after establishment. Some aquatic or sensitive species show growth penalties at high AgNP doses. (ResearchGate, ScienceDirect)

Safety and good practice

• NP quality & size matter: Smaller AgNPs are often more active (and more phytotoxic). Record supplier, size, coating. Keep light-protected stocks. (MDPI)
• Compatibility with autoclaving: Many labs add NPs after autoclaving and cooling (as a sterile bilayer or filter-sterilized addition) to avoid aggregation. (SpringerLink)
• Mercury-free goal: Where HgCl₂ is restricted, AgNPs and ZnO NPs are promising substitutes. Dispose of NP waste responsibly (follow institutional nanomaterial guidelines). (SpringerLink)

Table 1. Reported nanoparticle sterilization settings that worked in plant tissue culture

Species / system	NP & route	Trial dose & time	What improved	Notes
Rosa hybrida (nodal/shoot explants)	AgNP dip (surface)	200 mg/L, 20 min	Lower bacterial contamination; medium + 100 mg/L further reduced contamination & phenolic exudation	Multiple reports, similar settings in grapevine; confirm cultivar sensitivity. (ResearchGate, RSC Publishing)
Strawberry (Fragaria × ananassa)	AgNP dip; AgNP in medium	200 mg/L, 20 min better than 1 g/L HgCl₂; 0.5–1 mg/L in medium improved growth/ethylene	Faster rooting (+4 days), higher survival; lower ethylene in vessels	(e-services.nafosted.gov.vn)
Psidium friedrichsthalianum	AgNP bilayer on medium	5 mg/L on semisolid medium	Contamination down to ~40–50%; leaf area ↑ ~5.6×; multiplication ↑ ~1.8×	Add as sterile bilayer after pouring and inoculation. (SpringerLink)
Banana shoot tips	Zn or ZnO NPs in medium	100 mg/L	Achieved contamination-free cultures where bleach/HgCl₂ failed	Good option when endophytes persist. (RSC Publishing)
Barley callus	TiO₂ NPs in medium	~60 μg/mL	Eliminated bacterial contamination after a few subcultures	Monitor callus vigor. (RSC Publishing)
Limonium sinuatum	AgNP dip; AgNP in medium	200 mg/L, 20 min better than 1,000 mg/L HgCl₂, 5 min; ~1 mg/L in medium improved shoots	Also reduced ethylene; aided rooting at ~0.4 mg/L	(SpringerLink)
Scoparia dulcis (endophyte-rich)	Ag / Au / CuO NPs in medium (casein-stabilized)	~4 mg/L	Reduced endophytes; CuO strongest for regeneration	Titrate to avoid callus blackening at high dose. (Semantic Scholar)

Table 2. Pros & cons of NP-based sterilization

NP type	Pros	Watch-outs
AgNPs	Strong broad-spectrum action; effective as dip or low-dose in medium; may reduce ethylene and boost shoots	Phytotoxic at high doses (≥200 mg/L in some systems); species-specific responses; cost
Zn/ZnO NPs	Rescue when bleach/HgCl₂ fail (banana); good antibacterial activity	Typical doses (~100 mg/L) can stress sensitive tissues; check Zn toxicity
TiO₂ NPs	Work at very low μg/mL in callus systems	Light/photoreactivity, variable effects on morphogenesis
CuO NPs	Strong against endophytes in some medicinal plants	Narrow therapeutic window; high dose can blacken callus

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Step-by-step mini-protocol

1.     Prepare NP stock (e.g., AgNPs 1,000 mg/L). Store dark, sterile.

2.     Surface dip option: After routine ethanol rinse, immerse explants in AgNP 150–200 mg/L for 10–20 min. Rinse 2–3× with sterile water. Inoculate. (ResearchGate)

3.  Medium option: For persistent contamination, add AgNPs 1–5 mg/L (or ZnO ~100 mg/L) to cooled, autoclaved medium aseptically; for AgNPs you can also pipette a 5 mg/L bilayer on solidified medium with the explant already in place. (SpringerLink, RSC Publishing)

4.     Observe 2–3 weeks: Record contamination %, necrosis, chlorosis, shoot numbers, and time to rooting. Reduce or withdraw NPs if growth slows or leaves yellow. (ResearchGate)

Limitations & tips

• Not a magic bullet: Some aquatic or very soft tissues are NP-sensitive; NaOCl alone may be safer there. (ResearchGate)
• Mechanisms vary: NP size, coating, and plant species influence outcomes; always report these details in your lab notes/blog so others can reproduce. (MDPI)
• Green synthesis options: Plant/fungal-made AgNPs show antimicrobial activity, but characterization is essential for consistent results. (PMC)

MCQ on Sterilization in Plant Tissue Culture Using Nanoparticles

1. Which nanoparticle is most commonly reported for plant tissue culture sterilization?
A) Gold nanoparticles
B) Silver nanoparticles
C) Titanium dioxide nanoparticles
D) Copper oxide nanoparticles
Answer: B) Silver nanoparticles

2. The primary advantage of using AgNPs over mercuric chloride (HgCl₂) is:
A) Lower cost
B) No need for autoclaving
C) Mercury-free and less toxic to the environment
D) Stronger odor
Answer: C) Mercury-free and less toxic to the environment

3. In Rosa hybrida, surface sterilization with AgNPs was most effective at:
A) 50 mg/L for 5 min
B) 200 mg/L for 20 min
C) 500 mg/L for 2 min
D) 10 mg/L for 30 min
Answer: B) 200 mg/L for 20 min

4. Which metal oxide nanoparticles have been effective in rescuing banana shoot tips from persistent endophytes?
A) ZnO
B) TiO₂
C) Fe₂O₃
D) MgO
Answer: A) ZnO

5. What is a common method for adding AgNPs to the culture medium without aggregation?
A) Add before autoclaving
B) Add after autoclaving and cooling
C) Mix during pouring while hot
D) Freeze before adding
Answer: B) Add after autoclaving and cooling

6. Which of the following is a con of using nanoparticles in plant tissue culture?
A) Can reduce ethylene in culture vessels
B) May cause phytotoxicity at high doses
C) Can kill bacteria and fungi
D) Can replace HgCl₂
Answer: B) May cause phytotoxicity at high doses

7. In Psidium friedrichsthalianum, a bilayer of AgNPs at 5 mg/L on the medium resulted in:
A) Increased contamination
B) Decreased leaf area
C) Reduced contamination and increased multiplication rate
D) Slower rooting
Answer: C) Reduced contamination and increased multiplication rate

8. Which nanoparticle was reported to eliminate bacterial contamination in barley callus cultures at μg/mL levels?
A) Silver nanoparticles
B) Titanium dioxide nanoparticles
C) Zinc oxide nanoparticles
D) Copper oxide nanoparticles
Answer: B) Titanium dioxide nanoparticles

9. The broad-spectrum antimicrobial activity of nanoparticles is mainly due to:
A) Increasing pH of medium
B) Disrupting cell walls and releasing metal ions
C) Lowering oxygen levels in vessels
D) Adding plant hormones
Answer: B) Disrupting cell walls and releasing metal ions

10. Which of the following is not a recommended good practice when using nanoparticles in tissue culture?
A) Record NP size and coating
B) Use the lowest effective dose
C) Add directly to boiling medium
D) Dispose of NP waste responsibly
Answer: C) Add directly to boiling medium

References

1.     Ochatt et al. 2023. Application of nanoparticles in plant tissue cultures: minuscule size but huge effects. (review with sterilization examples). (SpringerLink)

2.   Rivera-Moreno et al. 2020. Argovit™ AgNPs reduce contamination and improve growth in vitro in Psidium friedrichsthalianum. Discover Applied Sciences. (bilayer 5 mg/L). (SpringerLink)

3.  RSC Review 2017. Nanomaterials in plant tissue culture: the disclosed and undisclosed. (Rosa hybrida 200 mg/L; Zn/ZnO rescue in banana; TiO₂ and others). (RSC Publishing)

4.     Shokri et al. (summarized in multiple sources). Rosa hybrida: 200 mg/L AgNP for 20 min reduced bacteria; 100 mg/L in medium reduced contamination and phenolics. (ResearchGate)

5.    SN Applied Sciences (open). Strawberry micropropagation: 200 mg/L AgNP dip outperformed 1 g/L HgCl₂; low in-medium AgNPs improved growth & reduced ethylene. (e-services.nafosted.gov.vn)

6.    Rakhimol et al. 2022–2023. Casein-stabilized Ag/Au/CuO NPs in Scoparia dulcis reduced endophytes; ~4 mg/L effective. (Semantic Scholar)

7.    Helaly et al. (reported in reviews). Zn or ZnO NPs (100 mg/L) solved persistent contamination in banana shoot tips. (RSC Publishing)

8.   Biosynthesis/AgNPs and contamination control in media (open-access overview with quantitative notes on 20–40 mg/L). (PMC)