May 13, 2026

Proposed Protocol for Agrobacterium-mediated Transformation of Tomato Cotyledons for HSP101

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l2dpppg1y/v1
  • Abdelrahman Budakhan Al Ali1
  • 1University of Queensland
Abdelrahman Budakhan Al Ali
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l2dpppg1y/v1
Protocol Citation: Abdelrahman Budakhan Al Ali 2026. Proposed Protocol for Agrobacterium-mediated Transformation of Tomato Cotyledons for HSP101 . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2dpppg1y/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Other
This proposed protocol is based on established principles of Agrobacterium-mediated plant transformation; however, the exact workflow has not yet been experimentally performed or optimized by the author. Therefore, successful transformation and regeneration are not guaranteed and must be validated under approved laboratory conditions.
Created: March 12, 2026
Last Modified: May 13, 2026
Protocol  Integer ID: 313143
Keywords: transformation of tomato cotyledon, protocol for agrobacterium, agrobacterium tumefacien, agrobacterium, ath-hsp101 gene, solanum lycopersicum, tomato cotyledon, camv 35s promoter, hsp101, promoter, tomato
Disclaimer
This protocol is a proposed educational workflow and has not been experimentally validated by the author. Any implementation must be conducted only in an approved laboratory under qualified supervision and in compliance with institutional biosafety and GMO regulations.
Abstract
This protocol describes the transformation of tomato (Solanum lycopersicum) cotyledons using Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pPBV[Exp]-Bar-(CaMV 35S)>ath_HSP101. The method allows stable integration of the ath_HSP101 gene under the CaMV 35S promoter 
Guidelines
This protocol is intended as a proposed educational and research-planning workflow for future laboratory validation. It has not yet been experimentally validated by the author. All experimental implementation should be conducted only in an approved laboratory under supervision of qualified personnel and in accordance with institutional biosafety procedures.

All plant tissue culture work should be performed using aseptic technique to reduce contamination. Media, instruments, culture vessels, and work surfaces should be sterilized before use. Explants should be handled gently to avoid excessive tissue damage, as this may reduce regeneration efficiency.

The transformation efficiency and regeneration response may vary depending on tomato cultivar, seedling age, explant condition, bacterial culture condition, antibiotic concentration, and plant growth regulator composition. Therefore, optimization may be required before successful transformation and regeneration can be achieved.

Putative transformed plants should not be considered confirmed transgenic plants until molecular confirmation has been completed. Suggested confirmation methods may include PCR-based detection of the inserted gene and/or additional molecular analysis where available.

All genetically modified plant material, bacterial cultures, and contaminated consumables should be handled, contained, and disposed of according to institutional biosafety and GMO waste-management requirements.
Materials
Biological materials
  • Solanum lycopersicum seedlings (7–10 days old)
  • Agrobacterium tumefaciens LBA4404 containing pPBV[Exp]-Bar-(CaMV 35S)>ath_HSP101
Reagents
  • LB broth
  • LB agar
  • Kanamycin
  • Cefotaxime
  • Murashige and Skoog (MS) medium
  • Agar
  • Sucrose
  • Acetosyringone
  • Silwet-L77
  • Sterile distilled water
Plant growth regulators
  • BAP (6-benzylaminopurine)
  • NAA (α-naphthaleneacetic acid)
  • IAA (indole-3-acetic acid)
Equipment
  • Orbital shaker incubator
  • Centrifuge
  • Laminar flow hood
  • Growth chamber or greenhouse
  • Petri dishes
  • Culture flasks
  • Autoclave

Safety warnings
This proposed protocol involves Agrobacterium tumefaciens, antibiotics, and genetically modified plant material. It must only be performed in an approved laboratory under proper biosafety supervision and in compliance with GMO regulations.
Ethics statement
This protocol does not involve human participants or animals. Therefore, human research ethics approval and animal ethics approval are not applicable.

However, this protocol involves genetically modified plant material and Agrobacterium tumefaciens. Before experimental implementation, approval should be obtained from the relevant institutional biosafety committee, GMO committee, or equivalent regulatory authority. All work should be conducted in accordance with local regulations for genetically modified organisms, plant biotechnology research, biological containment, and environmental safety.

If institutional approval is obtained in the future, the name of the approving committee, approval number, permit number, and approval date should be added to this protocol before experimental work begins.
Before start
Prepare all culture media and sterilize materials before beginning the experiment. Work under sterile conditions inside a laminar flow hood when handling plant tissues.
Ensure the following materials are ready:
  • Tomato seedlings (7–10 days old)
  • Agrobacterium tumefaciens LBA4404 carrying the HSP101 vector
  • LB media and antibiotics
  • Murashige and Skoog (MS) plant culture media
  • Plant growth regulators
Media preparation
LB broth (1 L)
Dissolve:
  • 10 g tryptone
  • 5 g yeast extract
  • 10 g NaCl
  • Adjust pH to 7.0.
  • Autoclave at 121 °C for 15–20 min.
Kanamycin stock solution
  • Dissolve 500 mg kanamycin in 10 mL sterile water.
  • Filter sterilize using a 0.22 µm filter.
  • Store at −20 °C.
  • Use 50 µg/mL final concentration in LB media.
Procedure
Preparation of Agrobacterium culture
  • Streak Agrobacterium tumefaciens LBA4404 on LB agar + kanamycin (50 µg/mL).
  • Incubate plates at 28 °C for 48–72 h.
  • Select a single colony and inoculate into 5–10 mL LB broth + kanamycin.
  • Incubate at 28 °C, 200–250 rpm for 16–24 h.
  • Transfer 1.5 mL of culture into 50 mL LB broth + kanamycin and incubate again until turbid.
Preparation of transformation suspension 
  • Centrifuge bacterial culture at 4000–5000 rpm for 10 min.
  • Discard the supernatant.
  • Resuspend the pellet in:
  1. 5% sucrose solution
  2. 0.05% Silwet-L77
Preparation of tomato cotyledon explants 
  • Germinate tomato seeds under sterile conditions.
  • After 7–10 days, excise cotyledons from seedlings.
  • Cut cotyledons into small sections to expose wounded tissue suitable for infection.
Infection of explants
  • Immerse cotyledon explants in the Agrobacterium suspension.
  • Incubate for 10–15 minutes with gentle agitation.
  • Remove explants and blot dry on sterile filter paper.
Co-cultivation
  • Transfer infected explants to co-cultivation medium containing:
  1. MS medium
  2. 100 µM acetosyringone
  • Incubate explants at 25 °C in darkness for 2–3 days.
Selection and shoot regeneration
  • Transfer explants to selection medium containing:
  1. MS medium
  2. 50 mg/L kanamycin
  3. 250 mg/L cefotaxime
  4. 3% sucrose
  5. 0.8% agar
  • Add plant growth regulators:
  1. 1 mg/L BAP
  2. 0.1 mg/L NAA
  • Incubate under 16 h light / 8 h dark photoperiod at 25 °C. (Shoots should emerge after 3–4 weeks.)
Root induction
  • Transfer regenerated shoots to rooting medium containing:
  1. ½ MS medium
  2. 0.5 mg/L IAA
  3. 3% sucrose
  • Roots typically develop within 1–2 weeks.
Acclimatization
  • Transfer rooted plants to soil pots.
  • Maintain plants in a greenhouse with controlled humidity.
  • Gradually acclimate plants to normal environmental conditions.