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Optimize Highly Efficient Genetic Transformation Method of Spring Wheat (Triticum aestivum L.) Cultivars Using Mature Embryo

Dongjin Kim Dongjin Kim
Megan Hager Megan Hager

Published: Nov 20, 2020 DOI: 10.21769/BioProtoc.3816 Views: 2274

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Abstract

Genetic engineering is a powerful tool to develop desired traits in crop plants to make the crops suitable for future demand and changing environments. For the genetic engineering of wheat cultivars, the development of efficient transformation and regeneration systems has always been a primary requirement. Immature embryos have been used as explants for callus generation and genetic transformation of wheat cultivars, but the availability of healthy immature embryos as an explant throughout the year is difficult. In contrast, mature embryos are relatively easy to use throughout the year. The following protocol describes the genetic transformation of wheat cultivars using a mature embryo as explants by the Agrobacterium-mediated transformation method.

Keywords: Wheat search Triticum aestivum search Transformation search Agrobacterium search Mature embryo search

Background

Genetic transformation is the most important technique used in wheat molecular genetics and improvement through genetic engineering. Microparticle bombardment and Agrobacterium-mediated transformation are the two most widely used methods for genetic transformation in wheat (Vasil et al., 1992 ; Ding et al., 2009). Agrobacterium-mediated transformation is a simple, low cost and highly efficient method compared to the direct gene delivery method. Hexaploid wheat possesses a large genome and shows a low rate of regeneration and transformation (Bhalla, 2006). Well-defined tissue culture procedures are needed for callus induction and regeneration for successful genetic engineering in crop plants. Immature embryos, mature embryos, and mature seedlings are used as explant sources. Among these, the most common source of explants is immature embryos due to their high transformation and regeneration efficiency. However, the wheat immature embryos are not readily available throughout the year, and there is a small window where the development stage of the immature embryo can be used for tissue culture (Alikina et al., 2016). When mature embryos are used as explants, they can be collected in large quantities and stored or used immediately. In this study, we used mature embryos from Montana wheat cultivars in order to optimize the protocol for the regeneration of callus and transformation using Agrobacterium-mediated methods.

Materials and Reagents

  1. 50 ml flask (Fisher Scientific, catalog number: 10-040C )

  2. 500 ml flask (PYREX, catalog number: 4980500 )

  3. Petri dish (Fisher Scientific, catalog number: FB0875713 )

  4. Wheat seeds

    Triticum aestivum L. cv Chinese Spring

    Triticum aestivum L. cv Amidon

    Triticum aestivum L. cv Choteau

  5. Agrobacterium tumefaciens strain LBA4404

  6. Rifampicin (Research Products International Corp, catalog number: R640001 )

  7. Streptomycin (Fisher BioReagents, catalog number: BP910-50 )

  8. Kanamycin (Fisher BioREagents, catalog number: BP906-5 )

  9. pGFPGUSPlus (Addgene, Plasmid, catalog number: 64401 )

  10. D-Mannitol (Sigma-Aldrich, catalog number: M4125 )

  11. L-glutamic acid (Spectrum Chemical, catalog number: GL125 )

  12. Yeast extract (Caisson Labs, catalog number: Y001 )

  13. Tryptone (Research Products International, catalog number: 60060 )

  14. KH2PO4 (Sigma-Aldrich, catalog number: 1551139 )

  15. NaCl (Sigma-Aldrich, catalog number: S9625 )

  16. MgSO4·7H2O (Sigma-Aldrich, catalog number: M2773 )

  17. MS basal salt (Bioworld, catalog number: 30630070-1 )

  18. D-Maltose (Caisson labs, catalog number: M004 )

  19. Casein hydrolysate (Sigma-Aldrich, catalog number: 22090 )

  20. Myo-inositol (Caisson labs, catalog number: M014 )

  21. L-Proline (Caisson labs, catalog number: P015 )

  22. Thiamine HCl (Caisson labs, catalog number: T009 )

  23. Copper(II) sulfate (Sigma-Aldrich, catalog number: C1297 )

  24. 2,4-Dichlorophenoxyacetic Acid (2,4-D) (Caisson labs, catalog number: D001 )

  25. X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) (GoldBio, catalog number: G1281 )

  26. Phytagel (Bioworld, catalog number: 40000035 )

  27. KCl (Sigma-Aldrich, catalog number: P9541 )

  28. Acetosyringone (Bioworld, catalog number: 40100297 )

  29. Timentin (Caisson labs, catalog number: T034 )

  30. Hygromycin (Spectrum Chemical, catalog number: H3399 )

  31. Indole-3-acetic Acid (IAA) (Caisson labs, catalog number: 1001 )

  32. 6-Benzylaminopurine (6-BAP) (Caisson labs, B001)

  33. Kinetin Hydrochloride (Spectrum Chemical, catalog number: K3010 )

  34. Lipoic Acid (spectrum Chemical, catalog number: L1515 )

  35. Ethanol (Sigma-Aldrich, catalog number: 16015 )

  36. Bleach (Clorox, catalog number: 65951360 )

  37. MG media (see Recipes)

  38. Agrobacterium resuspension medium (see Recipes)

  39. Co-cultivation media (see Recipes)

  40. Callus generation media (see Recipes)

  41. Resting media (see Recipes)

  42. Selection media (see Recipes)

  43. Regeneration media (see Recipes)

Equipment

  1. Zeiss AxioScope A1 (Carl Zeiss Microscopy, LLC USA)

  2. Plant growth chamber (Thermo Scientific, Precision 818 )

  3. Laminar airflow hood (Thermo Scientific, 1300 Series )

  4. Centrifuge (Eppendorf, 5804R )

  5. Shaker incubator (IKA works KS 4000i)

  6. Spectrophotometer (Eppendorf BioPhotometer, model: 6131 )

  7. Camera (Canon EOS Rebel T6)

Software

  1. ImageJ (NIH Image, webpage: imagej.net/ImageJ)

Procedure

Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant transformation > Agrobacterium

Plant Science > Plant cell biology > Tissue isolation and culture

Molecular Biology > DNA > Transformation

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