“Nanotechnology to Advance CRISPR–Cas Genetic Engineering of Plants” (Nature Nanotechnology, 2021)

🌱 Nanotechnology Meets CRISPR: A New Era for Smarter Crops

Feeding the world’s growing population while facing climate change is one of the greatest challenges of our time. To achieve this, scientists are turning to two powerful tools: CRISPR gene editing and nanotechnology. A recent paper published in Nature Nanotechnology explores how these two technologies can work together to revolutionize agriculture.

🌾 What is CRISPR and Why is it Important for Plants?

CRISPR–Cas is a cutting-edge genetic engineering tool that allows scientists to make precise changes in DNA. Unlike traditional breeding, which takes years and may introduce unwanted traits, CRISPR works like “molecular scissors,” targeting and editing only the desired genes.

In plants, CRISPR can:

• Improve resistance to drought, heat, and pests.
• Enhance nutrition in crops.
• Speed up the development of new plant varieties.

But while CRISPR is a game-changer, there’s a problem: delivering CRISPR tools into plant cells is very difficult. The plant cell wall acts like a strong protective shield, making it hard for the gene-editing machinery to get inside.

🔬 Enter Nanotechnology

This is where nanotechnology comes in. Nanoparticles tiny materials thousands of times smaller than a human hair can act as delivery vehicles for CRISPR components. Scientists have already shown that nanoparticles can carry DNA, RNA, and proteins into plant cells.

The big advantages of nanotechnology are:

• Efficient delivery of CRISPR tools across different plant species.
• Protection of fragile genetic material from breaking down.
• Targeted release inside specific plant tissues.
• Potential to reduce the need for complex and time-consuming tissue culture methods.

🚧 Challenges in Plant CRISPR Editing

Even with CRISPR, plant gene editing faces several hurdles:

1.     Cell Wall Barrier – Hard to cross without damaging the cell.

2.     Low Editing Efficiency – Success rates are still low in many crops.

3.     Species Limitations – Some plants respond better to editing than others.

4.     Regeneration Problem – Growing a whole plant from edited cells is slow and difficult.

5.     Regulatory Issues – Laws about genetically edited plants vary worldwide.

🌟 How Nanotech Can Help

The paper highlights how nanoparticles may solve these problems:

• Better Delivery: Nanoparticles can slip through the cell wall and reach the nucleus.
• DNA-Free Editing: They can deliver CRISPR proteins directly, reducing regulatory concerns.
• Species Independent: Since nanoparticles rely on physics, not plant biology, they may work across many crops.
• Boosting Efficiency: Smart nanoparticles could release CRISPR components at the right time and place, increasing success rates.
• Editing Reproductive Cells: Nanoparticles may even edit pollen or ovules, producing edited plants directly without tissue culture.

⚖️ Safety and Regulations

The paper also reminds us that safety matters. While nanotechnology is exciting, scientists must carefully study whether nanoparticles remain in plants, soil, or food after use. Regulations also differ:

• In the U.S., some CRISPR-edited crops are treated more lightly if no foreign DNA remains.
• In the EU, CRISPR plants face strict GMO regulations.
• Other countries like Brazil, Japan, and Australia have more flexible rules.

🌍 Why This Matters

Combining CRISPR with nanotechnology could:

• Help farmers grow crops that survive climate change.
• Reduce dependence on chemical fertilizers and pesticides.
• Speed up plant breeding for global food security.
• Support sustainable bioenergy and biomaterial production.

🧭 The Road Ahead

The researchers point out that many questions remain such as how much CRISPR cargo nanoparticles can carry, whether they can reach plant mitochondria or chloroplasts, and what long-term effects they may have. But the potential is enormous.

✨ Final Thought

Nanotechnology could be the missing link that makes CRISPR gene editing practical for all kinds of crops. If successful, this marriage of technologies may shape the future of farming, helping us feed billions of people sustainably.

📚 References

1.     Demirer, G. S., Silva, T. N., Jackson, C. T., Thomas, J. B., Ehrhardt, D. W., Rhee, S. Y., Mortimer, J. C., & Landry, M. P. (2021). Nanotechnology to advance CRISPR–Cas genetic engineering of plants. Nature Nanotechnology, 16(3), 243–250. https://doi.org/10.1038/s41565-021-00854-y

2.  Zhang, Y., Malzahn, A. A., Sretenovic, S., & Qi, Y. (2019). The emerging and uncultivated potential of CRISPR technology in plant science. Nature Plants, 5(8), 778–794.

3.     Zhu, H., Li, C., & Gao, C. (2020). Applications of CRISPR–Cas in agriculture and plant biotechnology. Nature Reviews Molecular Cell Biology, 21(11), 661–677.

4.  Cunningham, F. J., Goh, N. S., Demirer, G. S., Matos, J. L., & Landry, M. P. (2018). Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends in Biotechnology, 36(9), 882–897.

 

Biotechnology and Sustainable Agriculture: Shaping the Future of Farming

Introduction

Modern agriculture is facing a triple challenge: feeding a growing global population, minimizing environmental damage, and coping with climate change. Conventional practices such as excessive use of agrochemicals, monocropping, and over-exploitation of land are threatening soil fertility and biodiversity.

In this context, biotechnology has emerged as a powerful solution to create sustainable agricultural systems. A recent article published in PLOS Sustainability and Transformation highlights the potential of biotechnology in building eco-friendly, productive, and resilient farming systems (Das et al., 2023).

Importance of This Research

• Ensuring Food Security: Biotechnology can help address global food demands under conditions of drought, salinity, and emerging plant diseases.
• Reducing Agrochemical Use: Genetic engineering can create crops that naturally resist pests and diseases, reducing dependency on harmful pesticides.
• Nutritional Improvement: Biofortified crops (such as Vitamin-A enriched rice) can help combat hidden hunger and malnutrition.
• Environmental Protection: By lowering chemical use and preserving soil health, biotechnology supports ecological balance.

Key Roles of Biotechnology in Agriculture

1.     Crop Trait Enhancement

o    Development of high-yielding, pest-resistant, and climate-tolerant varieties.

o    Nutrient-rich crops that improve human health.

2.     Reducing Dependency on Chemicals

o    Example: Bt cotton produces its own insecticidal protein, minimizing pesticide use (Das et al., 2023).

3.     Faster Breeding & Micropropagation

o    Tissue culture allows rapid multiplication of disease-free and elite plant varieties.

o    Germplasm conservation ensures biodiversity protection.

4.     Improving Photosynthesis Efficiency

o    Genetic manipulation of photoprotection mechanisms in rice has enhanced photosynthetic efficiency and yield.

5.     Next-Generation Tools

o    CRISPR/Cas genome editing, OMICs technologies, and advanced gene mapping are revolutionizing crop improvement (Das et al., 2023).

Challenges Ahead

• Public Acceptance: Many consumers remain skeptical about genetically modified organisms (GMOs).
• Regulatory Hurdles: Different countries impose strict regulations on biotech crops, slowing down implementation.
• Equity Issues: Smallholder farmers may struggle to access advanced biotechnology unless policies ensure fair distribution.

Conclusion

Biotechnology is not just a laboratory innovation it is a practical solution for creating sustainable, resilient, and eco-friendly farming systems. By reducing chemical dependency, enhancing crop nutrition, and improving stress tolerance, biotechnology directly contributes to global food security and environmental protection.

However, responsible governance, public awareness, and equal access are essential for its success. If used wisely, biotechnology can pave the way for a green, sustainable, and food-secure future.

References

• Das, S., Ray, M. K., Panday, D., & Mishra, P. K. (2023). Role of biotechnology in creating sustainable agriculture. PLOS Sustainability and Transformation, 2(7), e0000069. https://doi.org/10.1371/journal.pstr.0000069

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)