Developing of transgenic wheat cultivars for improved disease resistance

A. H. Fahmy; R. A. Hassanein; H. A. Hashem; A. S. Ibrahim; O. M. El Shihy; E. A. Qaid

doi:10.7324/JABB.2018.60206

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Abstract

Wheat (Triticum aestivum L.) and (Triticum turgidum L.) are largely cultivated in the world. They are economically important because they can be grown in a wide range of climates and geographic regions. Wheat plants are exposed to a wide variety of disease pathogens, i.e., fungal, bacterial, and viral pathogens. The present work is an attempt to develop disease-resistant wheat cultivars. In this respect, immature embryo-derived calli of 2 wheat cultivars; Sids 1 (bread wheat) and Bani Suef 6 (durum wheat) were transformed by rice chitinase (cht 2), gus reporter gene, and selectable marker (bar) genes using particle bombardment technique. Transient gus expression in calli and stable gus expression in transformed nodes were observed. Transgenic calli were selected on phosphinothricin containing regeneration medium, and putative transformants were grown to maturity. 40 herbicide-resistant putative transformants were selected after leaf painting with 0.2% liberty herbicide. Presence and integration of transgenes were assessed by subjecting transgenic plants to polymerase chain reaction analysis using specific primers for gus, bar, and cht 2 genes. Transformation frequencies for cht 2 were 3.96% and 3.02% in Sids 1 and Bani Suef 6, respectively. The incorporation of rice chitinase gene in transformants was confirmed by Dot blot analyses.

Keyword: Rice chitinase Immature embryo Transformation Triticum aestivum Triticum durum.

Citation:

Fahmy AH, Hassanein RA, Hashem HA, Ibrahim AS, El Shihy OM, Qaid EA. Developing of transgenic wheat cultivars for improved disease resistance. J App Biol Biotech. 2018;6(2):31-40. DOI: 10.7324/JABB.2018.60206

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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Reference

1. Breiman A, Graur D. Wheat Evaluation. Israel J Plant Sci. 1995; 43: 58-95. https://doi.org/10.1080/07929978.1995.10676595
2. Bicar EH, Woodman-Clikeman W, Sangtong V, Peterson JM, Yang, SS, Lee M, Scott MP. Transgenic maize endosperm containing a milk protein has improved amino acid balance. Trans Res.2008; 17: 59-71. https://doi.org/10.1007/s11248-007-9081-3
3. Schlumbaum A, Mauch F, Vogeli U, Boller T. Plant chitinases are potent inhibitors of fungal growth. Nat.1986; 324: 365–367. https://doi.org/10.1038/324365a0
4. Leah R, Tommerup H, Svendsen I, Mundy J. Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J Biol Chem.1991; 266 (3): 1564-1573.
5. Velazhahan R, Samiyappan R, Vidhyasekaran P. Purification of an elicitor-inducible antifungal chitinase from suspension cultured rice cells. Phytoparasitica 2000; 28: 131–139. https://doi.org/10.1007/BF02981742
6. Wani SH. Inducing fungus resistance into plants through biotechnology. Not Sci Biol 2010; 2(2): 14-21.
7. Hamid R, Khan MA, Ahmad MM, Abdin MZ, Musarrat J, Javed A. Chitinase: An update. J Pharm Biol Sci 2013; 5 (1): 21-29.
8. Tabei Y, Kitade S, Nishizawa Y, Kikuchi N, Kayano T, Hibi T, Akutsu K. Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea). Plant Cell Rep 1998; 17: 159-164. https://doi.org/10.1007/s002990050371
9. Chen WP, Gu W, Liang GH, Muthukrishnan H, Chen PD, Liu DJ, Gill BS. Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker. Theor App Genetics 1998; 97: 1296– 1306. https://doi.org/10.1007/s001220051022
10. Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Mew TW, Muthukrishnan S, Datta SK. Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite Indica rice cultivars. Plant Sci 2001; 160(3): 405-414. https://doi.org/10.1016/S0168-9452(00)00413-1
11. Jabeen N, Chaudhary Z, Gulfraz M, Rashid H, Mirza B. Expression of Rice Chitinase Gene in Genetically Engineered Tomato Confers Enhanced Resistance to Fusarium Wilt and Early Blight. Plant Pathol J 2015; 31(3): 252–258. https://doi.org/10.5423/PPJ.OA.03.2015.0026
12. Fahmy AH, El-Shihy O, El-Shafey YH, Modkour MA. Genetic transformation of Egyptian wheat cultivar (Triticum aestivum L.) via biolistic bombardment using different constructs. Amer-Eurasian J Agri Environ Sci 2006; 1(2): 58-69.
13. Murashige T, Skoog F. A revised medium for rapid growth and Bioassays with tobacco tissue cultures. Physiol Plant 1962; 15: 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
14. Weeks JT, Anderson OD, Blechl AE. Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum L.). Plant Physiol 1993; 102: 1077–1084. https://doi.org/10.1104/pp.102.4.1077
15. El-Mangoury KA, Abdrabou RT, Yasien M, Fahmy AH. Optimization of a transformation system for three Egyptian wheat cultivars using immature embryo derived callus via micro-projectile bombardment. Arab J Biotech 2006; 9: 175-188.
16. Ausubel FM, Brent R, Kimgston RE, Moore D, Serdman JG, Smith A, Struhle K, Current protocols in molecular biology. Green Publishing Associates and Wiley Inter-science, New York, USA 1987; pp. 1-4.
17. Tu Z, He G, Li KX, Chen MJ, Chang J, Chen L, Yao Q, Li DP, Ye H, Shi J, Wu X. An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains. Electronic J Biotech 2005; 8(1):113-120.
18. Sanford JC, Smith FD, Russell JA. Optimizing the biolistic process for different biological application. Methods in Enzymology 1993; 217: 483-509. https://doi.org/10.1016/0076-6879(93)17086-K
19. Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 1987; 6: 3901–3907.
20. Cho MJ, Jiang W, Lemaux PO. Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci. 1998; 138: 229-244. https://doi.org/10.1016/S0168-9452(98)00162-9
21. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harboor, NY: Cold Spring Harbor Laboratory Press, 1989.
22. Vasil V, Castillo AM, Fromm ME, Vasil IK. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of re-generable embryogenic callus. Nat Biotech 1992; 10: 667–674. https://doi.org/10.1038/nbt0692-667
23. Nehra NS, Chibbar RN, Leung N, Caswell K, Mallard C, Steinhauer L, Baga M, Kartha KK. Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct geneconstructs. Plant J 1994; 5: 285–297. https://doi.org/10.1046/j.1365-313X.1994.05020285.x
24. Witrzens B, Brettell RI, Murray ER, McElroy D, Li Z, Dennis ES. Comparison of three selectable marker genes for transformation of wheat by microprojectile bombardment. Aust J Plant Physiol 1998; 25: 39–44. https://doi.org/10.1071/PP97095
25. Zhang L, Rybczynski JJ, Langenberg WG, Mitra A, French R. An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration. Plant Cell Rep 2000; 19: 241-250. https://doi.org/10.1007/s002990050006
26. Patnaik D, Khurana P. Genetic transformation of Indian bread (Triticum aestivum) and pasta (Triticum durum) wheat by particle bombardment of mature embryo-derived calli. BMC Plant Biol 2003; 3(5): 1-11.
27. Jia H, Yu J, Yi D, Cheng Y, Xu W, Zhang L, Ma Z. Chromosomal intervals responsible for tissue culture response of wheat immature embryos. Plant Cell Tissue Organ Cult 2009; 97(2): 159-165. https://doi.org/10.1007/s11240-009-9510-5
28. Y. He, H.D. Jones, S. Chen, X.M. Chen, D.W. Wang, K.X. Li, D.S. Wang, L.Q. Xia, Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency. J Exp Bot 2010; 61(6): 1567-1581. https://doi.org/10.1093/jxb/erq035
29. Fahmy AH, El Mangoury K, Abou El-Wafa W, Barakat HMS, El-Khodary S, Muthukrishnan S. Genetic transformation of Egyptian wheat (Triticum aestivum L.) with chitinase gene via microprojectile bombardment. Egyptian J Gene and Cyto 2013; 42: 233-245.
30. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K. Plant chitinases. The Plant J 1993; 3: 31–40. https://doi.org/10.1046/j.1365-313X.1993.t01-1-00999.x
31. Takahashi W, Fujimori M, Miura Y, Komatsu T, Nishizawa Y, Hibi T, Takamizo T. Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene. Plant Cell Rep 2005; 23: 811–818. https://doi.org/10.1007/s00299-004-0900-1
32. Shan XY, Li DS, Qu RD. Thidiazuron promotes in vitro regeneration of wheat and barley. In Vitro Cell Dev Biol- Plant 2006; 36 (3): 207- 210. https://doi.org/10.1007/s11627-000-0038-y
33. Ganeshan S, Chodaparambil SV, Ga MB, Fowler DB, Hucl P, Rossnagel PG, Chibbar RN. In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to thidiazuron. Plant Cell Tissue and Organ Cult 2006; 85: 63–73. https://doi.org/10.1007/s11240-005-9049-z
34. Gill AK, Gosal SS, Salu SK. Differential cultural responses of wheat (Triticum aestivum L.) with different explants. J Cell Tissue Res 2014; 14(2): 4351-4356.
35. Burdon V, Wickhan A, Lonsdale D, Harwood W. Additional introns inserted within the luciferase gene stabilize transgene expression in wheat. Plant Sci 2004; 167: 1143-1149. https://doi.org/10.1016/j.plantsci.2004.06.009
36. Tosi P, D'Ovidio R, Napier JA, Bekes PR, Expression of epitope-tagged LMW glutenin subunits in starchy endosperm of transgenic wheat and their incorporation into glutenin polymers. Theor Appl Gene 2004; 108(3): 468-476. https://doi.org/10.1007/s00122-003-1459-x
37. Machintosh CK, Lewis J, Radmer LE, Shin S, Heinen SJ, Smith LA, Wyckoff MN, Dill-Macky R, Evans CK, Kravchenko S, Baldridge GD, Zeyen RJ & Muehlbauerm GJ, Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight. Plant Cell Rep 2007; 26(4): 479-488. https://doi.org/10.1007/s00299-006-0265-8
38. Miroshnichenko DM, Poroshin GM, Dolgov SV. Genetic transformation of wheat using mature seed tissues. Appl Biochem Microbiol 2011; 47(8): 767-775. https://doi.org/10.1134/S0003683811080096
39 Brown T. Dot and slot blotting of DNA. In Current Protocols in Molecular Biology; John Wiley & Sons: Hoboken, NJ, USA, 2001. https://doi.org/10.1002/0471142727.mb0209bs21
40. Haddadi F, Abd Aziz M, Abdullah SNA, Tan SG, Kamaladini H. An Efficient Agrobacterium-Mediated transformation of strawberry cv. Camarosa by a dual plasmid system. Molecules 2015; 20: 3647-3666. https://doi.org/10.3390/molecules20033647
41. Becker D, Brettschneider R, Lorz H. Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J 1994; 5: 299-307. https://doi.org/10.1046/j.1365-313X.1994.05020299.x
42. Bieri S, Potrykus I, Fütterer I. Expression of active barley seed ribosome-inactivating protein in transgenic wheat. Theor App Gene 2000; 100: 755-763. https://doi.org/10.1007/s001220051349
43. Roy-Barman S, Sautter C, Chattoo BB. Expression of the lipid transfer protein Ace-AMP1 in transgenic wheat enhances antifungal activity and defense responses. Trans Res 2006; 5(4): 435-446. https://doi.org/10.1007/s11248-006-0016-1
44. Kasirajan L, Boomiraj K, Bansal KC. Optimization of genetic transformation protocol mediated by biolistic method in some elite genotypes of wheat (Triticum aestivum L.). Afri J Biotech 2013; 12(6): 531-538.

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Fahmy A H [PubMed] [Google Scholar ]

Hassanein R A [PubMed] [Google Scholar ]

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1. Breiman A, Graur D. Wheat Evaluation. Israel J Plant Sci. 1995; 43: 58-95. https://doi.org/10.1080/07929978.1995.10676595

2. Bicar EH, Woodman-Clikeman W, Sangtong V, Peterson JM, Yang, SS, Lee M, Scott MP. Transgenic maize endosperm containing a milk protein has improved amino acid balance. Trans Res.2008; 17: 59-71. https://doi.org/10.1007/s11248-007-9081-3

3. Schlumbaum A, Mauch F, Vogeli U, Boller T. Plant chitinases are potent inhibitors of fungal growth. Nat.1986; 324: 365–367. https://doi.org/10.1038/324365a0

4. Leah R, Tommerup H, Svendsen I, Mundy J. Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J Biol Chem.1991; 266 (3): 1564-1573.

5. Velazhahan R, Samiyappan R, Vidhyasekaran P. Purification of an elicitor-inducible antifungal chitinase from suspension cultured rice cells. Phytoparasitica 2000; 28: 131–139. https://doi.org/10.1007/BF02981742

6. Wani SH. Inducing fungus resistance into plants through biotechnology. Not Sci Biol 2010; 2(2): 14-21.

7. Hamid R, Khan MA, Ahmad MM, Abdin MZ, Musarrat J, Javed A. Chitinase: An update. J Pharm Biol Sci 2013; 5 (1): 21-29.

8. Tabei Y, Kitade S, Nishizawa Y, Kikuchi N, Kayano T, Hibi T, Akutsu K. Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea). Plant Cell Rep 1998; 17: 159-164. https://doi.org/10.1007/s002990050371

9. Chen WP, Gu W, Liang GH, Muthukrishnan H, Chen PD, Liu DJ, Gill BS. Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker. Theor App Genetics 1998; 97: 1296– 1306. https://doi.org/10.1007/s001220051022

10. Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Mew TW, Muthukrishnan S, Datta SK. Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite Indica rice cultivars. Plant Sci 2001; 160(3): 405-414. https://doi.org/10.1016/S0168-9452(00)00413-1

11. Jabeen N, Chaudhary Z, Gulfraz M, Rashid H, Mirza B. Expression of Rice Chitinase Gene in Genetically Engineered Tomato Confers Enhanced Resistance to Fusarium Wilt and Early Blight. Plant Pathol J 2015; 31(3): 252–258. https://doi.org/10.5423/PPJ.OA.03.2015.0026

12. Fahmy AH, El-Shihy O, El-Shafey YH, Modkour MA. Genetic transformation of Egyptian wheat cultivar (Triticum aestivum L.) via biolistic bombardment using different constructs. Amer-Eurasian J Agri Environ Sci 2006; 1(2): 58-69.

13. Murashige T, Skoog F. A revised medium for rapid growth and Bioassays with tobacco tissue cultures. Physiol Plant 1962; 15: 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

14. Weeks JT, Anderson OD, Blechl AE. Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum L.). Plant Physiol 1993; 102: 1077–1084. https://doi.org/10.1104/pp.102.4.1077

15. El-Mangoury KA, Abdrabou RT, Yasien M, Fahmy AH. Optimization of a transformation system for three Egyptian wheat cultivars using immature embryo derived callus via micro-projectile bombardment. Arab J Biotech 2006; 9: 175-188.

16. Ausubel FM, Brent R, Kimgston RE, Moore D, Serdman JG, Smith A, Struhle K, Current protocols in molecular biology. Green Publishing Associates and Wiley Inter-science, New York, USA 1987; pp. 1-4.

17. Tu Z, He G, Li KX, Chen MJ, Chang J, Chen L, Yao Q, Li DP, Ye H, Shi J, Wu X. An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains. Electronic J Biotech 2005; 8(1):113-120.

18. Sanford JC, Smith FD, Russell JA. Optimizing the biolistic process for different biological application. Methods in Enzymology 1993; 217: 483-509. https://doi.org/10.1016/0076-6879(93)17086-K

19. Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 1987; 6: 3901–3907.

20. Cho MJ, Jiang W, Lemaux PO. Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci. 1998; 138: 229-244. https://doi.org/10.1016/S0168-9452(98)00162-9

21. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harboor, NY: Cold Spring Harbor Laboratory Press, 1989.

22. Vasil V, Castillo AM, Fromm ME, Vasil IK. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of re-generable embryogenic callus. Nat Biotech 1992; 10: 667–674. https://doi.org/10.1038/nbt0692-667

23. Nehra NS, Chibbar RN, Leung N, Caswell K, Mallard C, Steinhauer L, Baga M, Kartha KK. Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct geneconstructs. Plant J 1994; 5: 285–297. https://doi.org/10.1046/j.1365-313X.1994.05020285.x

24. Witrzens B, Brettell RI, Murray ER, McElroy D, Li Z, Dennis ES. Comparison of three selectable marker genes for transformation of wheat by microprojectile bombardment. Aust J Plant Physiol 1998; 25: 39–44. https://doi.org/10.1071/PP97095

25. Zhang L, Rybczynski JJ, Langenberg WG, Mitra A, French R. An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration. Plant Cell Rep 2000; 19: 241-250. https://doi.org/10.1007/s002990050006

26. Patnaik D, Khurana P. Genetic transformation of Indian bread (Triticum aestivum) and pasta (Triticum durum) wheat by particle bombardment of mature embryo-derived calli. BMC Plant Biol 2003; 3(5): 1-11.

27. Jia H, Yu J, Yi D, Cheng Y, Xu W, Zhang L, Ma Z. Chromosomal intervals responsible for tissue culture response of wheat immature embryos. Plant Cell Tissue Organ Cult 2009; 97(2): 159-165. https://doi.org/10.1007/s11240-009-9510-5

28. Y. He, H.D. Jones, S. Chen, X.M. Chen, D.W. Wang, K.X. Li, D.S. Wang, L.Q. Xia, Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency. J Exp Bot 2010; 61(6): 1567-1581. https://doi.org/10.1093/jxb/erq035

29. Fahmy AH, El Mangoury K, Abou El-Wafa W, Barakat HMS, El-Khodary S, Muthukrishnan S. Genetic transformation of Egyptian wheat (Triticum aestivum L.) with chitinase gene via microprojectile bombardment. Egyptian J Gene and Cyto 2013; 42: 233-245.

30. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K. Plant chitinases. The Plant J 1993; 3: 31–40. https://doi.org/10.1046/j.1365-313X.1993.t01-1-00999.x

31. Takahashi W, Fujimori M, Miura Y, Komatsu T, Nishizawa Y, Hibi T, Takamizo T. Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene. Plant Cell Rep 2005; 23: 811–818. https://doi.org/10.1007/s00299-004-0900-1

32. Shan XY, Li DS, Qu RD. Thidiazuron promotes in vitro regeneration of wheat and barley. In Vitro Cell Dev Biol- Plant 2006; 36 (3): 207- 210. https://doi.org/10.1007/s11627-000-0038-y

33. Ganeshan S, Chodaparambil SV, Ga MB, Fowler DB, Hucl P, Rossnagel PG, Chibbar RN. In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to thidiazuron. Plant Cell Tissue and Organ Cult 2006; 85: 63–73. https://doi.org/10.1007/s11240-005-9049-z

34. Gill AK, Gosal SS, Salu SK. Differential cultural responses of wheat (Triticum aestivum L.) with different explants. J Cell Tissue Res 2014; 14(2): 4351-4356.

35. Burdon V, Wickhan A, Lonsdale D, Harwood W. Additional introns inserted within the luciferase gene stabilize transgene expression in wheat. Plant Sci 2004; 167: 1143-1149. https://doi.org/10.1016/j.plantsci.2004.06.009

36. Tosi P, D'Ovidio R, Napier JA, Bekes PR, Expression of epitope-tagged LMW glutenin subunits in starchy endosperm of transgenic wheat and their incorporation into glutenin polymers. Theor Appl Gene 2004; 108(3): 468-476. https://doi.org/10.1007/s00122-003-1459-x

37. Machintosh CK, Lewis J, Radmer LE, Shin S, Heinen SJ, Smith LA, Wyckoff MN, Dill-Macky R, Evans CK, Kravchenko S, Baldridge GD, Zeyen RJ & Muehlbauerm GJ, Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight. Plant Cell Rep 2007; 26(4): 479-488. https://doi.org/10.1007/s00299-006-0265-8

38. Miroshnichenko DM, Poroshin GM, Dolgov SV. Genetic transformation of wheat using mature seed tissues. Appl Biochem Microbiol 2011; 47(8): 767-775. https://doi.org/10.1134/S0003683811080096

39 Brown T. Dot and slot blotting of DNA. In Current Protocols in Molecular Biology; John Wiley & Sons: Hoboken, NJ, USA, 2001. https://doi.org/10.1002/0471142727.mb0209bs21

40. Haddadi F, Abd Aziz M, Abdullah SNA, Tan SG, Kamaladini H. An Efficient Agrobacterium-Mediated transformation of strawberry cv. Camarosa by a dual plasmid system. Molecules 2015; 20: 3647-3666. https://doi.org/10.3390/molecules20033647

41. Becker D, Brettschneider R, Lorz H. Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J 1994; 5: 299-307. https://doi.org/10.1046/j.1365-313X.1994.05020299.x

42. Bieri S, Potrykus I, Fütterer I. Expression of active barley seed ribosome-inactivating protein in transgenic wheat. Theor App Gene 2000; 100: 755-763. https://doi.org/10.1007/s001220051349

43. Roy-Barman S, Sautter C, Chattoo BB. Expression of the lipid transfer protein Ace-AMP1 in transgenic wheat enhances antifungal activity and defense responses. Trans Res 2006; 5(4): 435-446. https://doi.org/10.1007/s11248-006-0016-1

44. Kasirajan L, Boomiraj K, Bansal KC. Optimization of genetic transformation protocol mediated by biolistic method in some elite genotypes of wheat (Triticum aestivum L.). Afri J Biotech 2013; 12(6): 531-538.