- Home
- Protocols
-
Barley Transformation by Particle Bombardment Using Callus
Published: Jun 5, 2020 DOI: 10.21769/BioProtoc.3632 Views: 2619
Edited by: Samik Bhattacharya Reviewed by: Tohir Bozorov
Download PDF
Ask a question
Abstract
The delivery of desired construct into plant cells relies mainly on Agrobacterium-mediated transformation. However, Agrobacterium transformation is still very challenging for barley as only one genotype is amendable to Agrobacterium infection. The demand for developing genotype independent transformation methods is thus high. Here we report a transformation protocol for barley cultivar “Rika” by particle bombardment.
Keywords: Barley
Particle bombardment
Immature embryo
Transformation
Background
Tissue culture based Agrobacterium transformation is commonly used for introducing vector DNAs into plant cells, but it is often genotype dependent. The availability of an efficient transformation protocol is thus crucial for the success of genome editing via Agrobacterium transformation. Barley is one of the economically important cereal crops, unfortunately, Agrobacterium transformation is in principle only working for the cultivar ’Golden Promise’. Developing alternative transformation methods is thus required to facilitate genetic improvement by genome editing of the crop. Particle bombardment can deliver vector DNAs into plant cells directly, which could be an alternative transformation method for barley. Here we report a particle bombardment protocol for barley transformation modified according to wheat particle bombardment using callus (Tian et al., 2019).
Materials and Reagents
- 0.6 μm gold particle (Bio-Rad, catalog number: 1652262 )
- Macrocarrier disk (Bio-Rad, catalog number: 1652335 )
- Stopping screen (Bio-Rad, catalog number: 1652336 )
- 1,350 psi rupture disk (Bio-Rad, catalog number: 1652330 )
- Micropore tape (3M, catalog number: 1530-1 )
- Petri dish
- Eppendorf tube
- Barley cv. Rika seeds
- Calcium chloride (Sigma-Aldrich, catalog number: C1016-100G )
- Sodium hypochlorite, 6-14% (Sigma-Aldrich, catalog number: 425044-250ML )
- Spermidine (Sigma-Aldrich, catalog number: S2626 )
- Isopropanol (Sigma-Aldrich, catalog number: I19516-500ML )
- MS medium (Duchefa, catalog number: M0245 )
- Casein hydrolysate (Duchefa, catalog number: C1301 )
- CuSO4·5H2O (Sigmal-Aldrich, catalog number: 451657-10G )
- Sucrose (Duchefa, catalog number: S0809 )
- Maltose (Duchefa, catalog number: M0811 )
- Glutamine (Duchefa, catalog number: G0708 )
- Proline (Duchefa, catalog number: P0717 )
- Dicamba (Duchefa, catalog number: D0920 )
- Ascorbic acid (Duchefa, catalog number: A0602 )
- Zeatin riboside (Duchefa, catalog number: Z0937 )
- Gelrite (Duchefa, catalog number: G1101 )
- Ethanol
- Plasmid DNA (see Recipes)
- Gold particle stock solution (see Recipes)
- Calcium chloride stock solution (2.5 M, CaCl2) (see Recipes)
- Spermidine stock solution (0.1 M) (see Recipes)
- Callus induction medium (see Recipes)
- Shoot induction medium (see Recipes)
- Potassium hydroxide solution (1 M) (see Recipes)
Equipment
- Biolistic PDS-1000/He Particle delivery system (Bio-Rad, model: 165-2257 )
- Welch ILMVAC Rotary vane vacuum pump (Fisher Scientific)
- Helium gas tank (AGA, Sweden).
- Centrifuge (Eppendorf, model: 5417R )
- Stereo microscope (Olympus)
- Climate chamber
- Laminar flow hood
Procedure
Category
Plant Science > Plant transformation > Bombardment
Plant Science > Plant molecular biology > DNA
Molecular Biology > DNA > Transformation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Post a Question
