May 07, 2026
An efficient wheat tissue culture regeneration protocol from mature embryos
- Manavi Raizada1,
- Vandan Patila1,
- Ramawatar Nagar1
- 1ICAR-National Institute for Plant Biotechnology
- Plant transformation and genome editing
Protocol Citation: Manavi Raizada, Vandan Patila, Ramawatar Nagar 2026. An efficient wheat tissue culture regeneration protocol from mature embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkzqb5l5r/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: In development
We are still developing and optimizing this protocol
Created: April 24, 2026
Last Modified: May 07, 2026
Protocol Integer ID: 315649
Keywords: Wheat , Triticam astivam, transformation, regeneration, mature embryo, efficient wheat tissue culture regeneration protocol, wheat transformation, explants for wheat transformation, plant regeneration, wheat genome, mature embryos despite advance, limited seasonal availability of immature embryo, high regeneration capacity, mature embryo, physiological state of mature embryo, convenient alternative for regeneration, maintaining donor plant, wheat, complexity of the wheat genome, regeneration, donor plant, immature embryo, specific harvesting requirement
Abstract
Despite advances in wheat transformation, plant regeneration and transformation efficiency remain low, mainly due to the complexity of the wheat genome and genotype-dependent responses. Although immature embryos are the preferred explants for wheat transformation because of their high regeneration capacity, maintaining donor plants is expensive, time-consuming, and labour-intensive. In addition, the stage-specific harvesting requirement and limited seasonal availability of immature embryos further constrain their use. In contrast, mature embryos represent a more convenient alternative for regeneration and transformation because they are available year-round and can be easily isolated. Moreover, the physiological state of mature embryos shows relatively low variability
Image Attribution
Images were taken by Vandan Patial and MImagesanavi Raizada
Materials
Biological Materials: Mature wheat seeds of Indian genotype as explant.
Chemical Materials: double distilled water, triton X, 1% bavistin, sodium hypochlorite (4%) solution, 70 % ethanol, Murashige and Skoog medium (Duchefa - M0222), 2, 4-D (Sigma Aldrich – D7299), zeatin (Duchefa – Z0917), casein hydrolysate, proline, asparagine, copper sulphate, sucrose, agar agar
Glassware: test tubes, jam bottles, conical flasks, beakers, scott bottles
Other Materials: forceps, scalpel, scissors, petri plates, test tube stand, filter paper, cotton, blotting sheet, parafilm, burner/ lamp, matchbox/ lighter, micropipette and its tips, soilrite, pots, clear plastic bags
Troubleshooting
Problem
Precocious Germination
Solution
remove the embryonic axis 2-3 days post inoculation, with the help of a magnifying lamp.
Before start
- Take properly stored healthy seeds as starting material.
- Adjust pH of all media to 5.8 before autoclaving.
- Autoclave all possible materials.
Surface sterilization & soaking of seeds
35m
Seeds were washed for 10 minutes with tap water having 2-3 drops of triton X with continuous stirring.
10m
This was followed by wash them with running tap water for 5-10 minutes.
5m
Seeds were then treated with 1% Bavistin for 1-2 minutes to avoid seed born fungal infection.
2m
Seeds were rinsed 2-3 times in laminar airflow (LAF) with autoclaved double distilled water to remove excess fungicide.
2m
After this, seeds were treated with 70% ethanol for 2 minutes
2m
Follow by surface sterilization using 4% sodium hypochlorite for 10-12 minutes.
10m
Surface sterilized seeds were rinsed 3-5 times with autoclaved double distilled water to remove excess of sodium hypochlorite.
4m
These sterilized seeds were then left to soak overnight inside the LAF in a sealed petri plate at room temperature.
Embryo excision
Intact mature embryos (between 2-4 mm in size) were extracted aseptically using fine forceps and scalpel in laminar airflow with the help of a USB desk lamp & magnifier, and kept on sterilized petri plate layered with autoclaved wet filter paper.
Extracted embryos, with scutellum side up, were inoculated on callus induction medium for callogenesis, around 30 per plate. Petri plates were sealed with parafilm and incubated at 24 ± 2°C in the dark.
Extraction of embryos should be done carefully, taking special care to avoid extracting extra tissue along with the embryo.
Callus induction medium
MS (Murashige and Skoog medium)(4.3 g/l) +2, 4-D (2mg/l) + casein hydrolysate (1g/l) + proline (0.6 g/l) + asparagine (50mg/l) + sucrose (3%) + agar (0.8%)
Embryos excised on Callus Induction media scutellum side up.
Shoot regeneration
Embryogenic calli were split into smaller size and transferred to regeneration medium.
Cultures were incubated under light at 24 ± 2°C with 16 hours photoperiod. Shoot regeneration started after 6-7 days of inoculation.
Shoot regeneration medium
MS (4.3 g/l) + zeatin (5 mg/l) + copper sulphate (3 mg/l) + casein hydrolysate (1 g/l) + proline (0.6 g/l) + asparagine (50 mg/l) + sucrose (3%) + agar (0.8%)
shoot induction on regeneration media
Rhizogenesis
Regenerated shoots (1-2 cm long) were transferred to 1/2 MS medium for rooting.
Rhizogenesis medium
1/2 MS+ sucrose (3%) + agar (0.8%)
Regenerated plant with developed roots
Acclimatization
After 15-20 days, well-rooted plantlets were gently removed from medium using long forceps.
Roots were washed with lukewarm water to clear any tissue culture medium left.
Care should be taken while removing the in vitro grown plantlets from medium before shifting to soilrite.
In vitro regenerated plantlets were transplanted to pots containing sterilized soilrite and covered with clear plastic bags to maintain high humidity while the plants become established in soil. These plantlets were placed in the growth chamber at 21-22⁰ C with 16 hrs photoperiod.
After 1-week, plastic bags were removed. Hardened plants were grown under standard green house conditions until maturity.
Wheat regenerated plants in soilrite for acclimatization