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

Abstract

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

1. Seed sterilization

   1. Select healthy seeds and soak in sterile distilled water for 30 min (after these steps are done in laminar airflow hood).

   2. Treat with 70% ethanol (v/v) for 5 min, and wash with sterile distilled water for three times.

   3. Sterile seed with 30% (v/v) commercial bleach in shaking incubator at room temperature for 30 min.

   4. Finally, wash seeds 5-6 times with sterile water.

      
      

2. Mature embryo preparation and callus induction

   1. After surface sterilization, germinate seeds overnight on moist filter paper at 25 °C in the dark.

   2. Excise the embryos from the endosperm using forceps and a fine scalpel under a laminar hood.

   3. Place the 20-30 embryos scutellum facing up on the callus generation media.

   4. Incubate the embryos at 25 °C in the dark for 2 weeks.

   5. After two weeks, transfer only embryogenic callus to fresh callus regeneration media.

   6. Keep the embryogenic callus at 25 °C incubator in the dark for 4 weeks prior to transformation.

      
      

      
      Figure 1. Callus generation step from mature seed. Over-night incubation of mature seeds on moist filter paper at 25 °C incubator in the dark. The next day, extract embryo using forceps and a fine scalpel under a laminar hood. Place the embryos on the callus generation media and incubate in the dark at 25 °C. Observed callus after 3 days on the callus generation media, and separate embryogenic callus from the embryo after 10 days.

      
      

3. Preparation of Agrobacterium cells and co-cultivation with wheat callus

   1. Incubate a single colony of Agrobacterium strain LBA4404 in 10 ml MG media supplemented with Rifampicin, Streptomycin and Kanamycin in 50 ml flask overnight at 28 °C on shaking incubator at 250 rpm.

   2. The next day, add 1% of the primary culture to a 500 ml flask containing 100 ml MG medium.

   3. Cultivate the cells to reach OD₆₀₀ > 1 (optical density). Normally, it takes 16-18 h.

   4. Pellet the Agrobacterium culture at 12,000 rpm for 15 min at 4 °C.

   5. Resuspend the pellet with the Agrobacterium resuspension medium to adjust the OD₆₀₀ to 0.3.

   6. Place the 4-week-old 25-30 embryogenic callus into a sterile Petri dish.

   7. Pour the 100 µl of resuspended Agrobacterium on the embryogenic calli, and incubate the Agrobacterium on the calli for 5 min.

   8. Transfer the inoculated calli to sterile filter paper and dry for 5 min.

   9. Transfer the calli to the co-cultivation medium and culture the callus for 3 days at 24 °C in the dark.

      
      

      
      Figure 2. The co-cultivation procedure of the embryogenic callus and Agrobacterium. Pour the 100 µl resuspended Agrobacterium on the 4-week-old embryogenic calli and incubation the callus for 5 min. Transfer the callus on the sterilized filter paper and dry 5 min. Transfer the callus to the co-cultivation medium.

      
      

4. Selection and regeneration of transgenic plant

   1. Rinse the calli 5-6 times with 160 mg/L timentin solution and dry the calli on sterilized filter paper for 5 min.

   2. Transfer the dried calli onto resting media for 10 days at 24 °C in the dark.

   3. After 10 days of resting, transfer the calli to the selection medium for 2 weeks at 25 °C in the dark.

   4. Remove brown and black calli from the selection medium and transfer healthy calli to the fresh section media for the second selection.

   5. Incubate the selected calli at 25 °C under low-light intensity (Normal plant growth condition is approx.130-150 μE m^-2 s^-1 light intensity, but in this step, cover the calli with one sheet of white paper for reduced light intensity).

   6. After 10 days on the second selection, transfer healthy calli onto regeneration medium at 25 °C under 16 h light/8 h dark condition.

   7. Every 10-day transfer calli to fresh regeneration medium up to 6 weeks until roots and shoots are observed.

   8. Continue transferring calli and plantlets to fresh selection media for shoot regeneration.

   9. Transfer shoot regenerated plantlets to Magenta vessels having MS media without any hormone.

      
      

5. Analysis of the regenerated transgenic callus and plants

   1. GFP expression test.

      1. GFP expression is detected in the callus 3 days after co-cultivation with Agrobacterium, using a Zeiss Axio scope A1 (Carl Zeiss Microscopy, LLC USA).

      2. Set-up the microscope with GFP filter, 470/40 nm excitation filter.

      3. Observe at least 20 individual callus under the microscope and count GFP expressed cell number.

   2. GUS activity assay with leaf segment.

      1. Cut the 1 cm leaf of each regenerated plantlet from the root generation media (Magenta vessels) and place the leaf fragments into the microplate with 100 μl X-Gluc solution.

      2. Incubate the plate at 37 °C overnight (color to fully develop).

      3. After incubation, remove the chlorophyll in leaf fragments by replacing X-gluc with 70% ethanol under the light until the green color fades.

      4. The blue-stained product is visible.

Data analysis

1. Callus induction frequency on the mature embryos

   1. Three different genotypes (Triticum aestivum L. cv Chinese Spring, Triticum aestivum L. cv Amidon, and Triticum aestivum L. cv Choteau) of spring wheat examine for callus induction and plant regeneration.

   2. 50-60 s of each genotype examine callus induction frequency in each replication.

   3. After 2 weeks on the callus generation media, determine callus induction rate by size of the callus on the embryos and callus development monitor for 4 weeks.

   4. Take a picture of the callus generation plates and measure the longest length of callus by using the ImageJ program.

   5. Determine callus induction by over 5 mm of callus length. And shorter than 5 mm of callus keep growing on the callus generation media for 2 more weeks.

   6. Callus induction frequency calculate as the number of calli generated embryo divide by the total number of embryos on the plate multiply 100.

      
      

      

      
      

      
      Figure 3. Measure the length of the callus. The yellow line measures the most extended length of the calli using the ImageJ program, and the scale bar is 5 mm. Measure the callus size for determining the callus generation frequency.

      
      

2. The efficiency of the transgenic plant regeneration

   1. After 4 weeks from the co-cultivation, start appearing a green tissue on the surface of the callus and generating a shoot on the green tissue.

   2. When a shoot generates on the callus, individual shoots separate from the regeneration calli and place in a Magenta vessel having MS media without growth hormone.

   3. Count the number of shoots out of the total number of transformed callus.

   4. Shoot generation rate is
      
      

      
      

   5. Keep growing for 2 weeks more and count the number of shoots and calculate the final shoot generation rate.

   6. After 4 weeks in the Magenta vessels, count the root induction rate.

   7. Root induction rate is

      
      
      
      

   8. The root induction rate is the same as the plant regeneration rate.

Recipes

1. MG media

   5 g/L Mannitol

   1 g/L L-glutamic acid

   2.5 g/L Yeast extract

   5 g/L tryptone

   250 mg/L KH₂PO₄

   100 mg/L NaCl

   100 mg/L MgSO₄·7H₂O

   pH 7.0

   Add supplement after autoclaving

   1 μg/L Biotin

   25 mg/L Streptomycin

   10 mg/L Rifampicin

   50 mg/L Kanamycin

2. Agrobacterium resuspension medium

   4.3 g/L MS salts

   30 g/L maltose

   3 g/L KCl

   4 g/L MgCl₂

   150 µM Acetosyringone

   pH 5.2

3. Co-cultivation media

   4.3 g/L MS basal salt

   1 g/L Casein hydrolysate

   350 mg/L Myo-inositol

   690 mg/L Proline

   1 mg/L Thiamine HCl

   5 pM copper (II) sulfate

   150 µM Acetosyringone

   0.7% Phytagel

   pH 5.8

4. Callus generation media

   4.3 g/L MS basal salt

   1 g/L Casein hydrolysate

   350 mg/L Myo-inositol

   690 mg/L Proline

   1 mg/L Thiamine HCl

   5 pM Copper(II) sulfate

   2.5 mg/L 2,4-D

   0.7% Phytagel

   pH 5.8

5. Resting media

   4.3 g/L MS basal salt

   1 g/L Casein hydrolysate

   350 mg/L Myo-inositol

   690 mg/L Proline

   1 mg/L Thiamine HCl

   5 pM Copper(II) sulfate

   2.5 mg/L 2,4-D

   160 mg/L Timetine

   0.7% Phytagel

   pH 5.8

6. Selection media

   4.3 g/L MS basal salt

   1 g/L Casein hydrolysate

   350 mg/L Myo-inositol

   690 mg/L Proline

   1 mg/L Thiamine HCl

   5 pM Copper(II) sulfate

   160 mg/L Timetine

   2 mg/L Lipoic acid

   1 µM IAA

   25 mg/L Hygromycin

   0.7% Phytagel

   pH 5.8

7. Regeneration Media

   4.3 g/L MS basal salt

   1 g/L Casein hydrolysate

   350 mg/L Myo-inositol

   690 mg/L Proline

   1 mg/L Thiamine HCl

   5 pM Copper(II) sulfate

   160 mg/L Timetine

   1 mg/L 6-BAP

   1 mg/L Kinetin

   25 mg/L Hygromycin

   0.7% Phytagel

   pH 5.8

Acknowledgments

This work was supported by a grant from the Montana Wheat and Barley Committee.

Competing interests

The authors declare no competing interest.

References

1. Alikina, О., Chernobrovkina, M., Dolgov, S. and Miroshnichenko, D. (2016). Tissue culture efficiency of wheat species with different genomic formulas. Crop Breed Appl Biotechnol 16(4): 307-314.
2. Bhalla, P. L., (2006). Genetic engineering of wheat--current challenges and opportunities. Trends Biotechnol 24(7): 305-311.
3. Ding, L., Li, S., Gao, J., Wang, Y., Yang, G. and He, G. (2009). Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Mol Biol Rep 36(1): 29-36.
4. Vasil, V., Castillo, A. M., Fromm, M. E. and Vasil, I. K. (1992). Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Nature Biotechnol 10(6): 667-674.