Advertisement

Agrobacterium-mediated transformation of alfalfa (Medicago sativa) using a synthetic cry3a gene to enhance resistance against alfalfa weevil

  • Masoud TohidfarEmail author
  • Naser Zare
  • Gholamreza Salehi Jouzani
  • Seide Maryam Eftekhari
Original Paper

Abstract

To introduce genetic resistance against alfalfa weevil (Hypera postica), leaves and petiole explants of three commercial alfalfa genotypes, including Km-27, Kk-14 and Syn-18 were transformed with Agrobacterium tumefaciens strains GV101, LBA4404 and AGL01. All the Agrobacterium strains used harbored the recombinant binary vector pBI121 containing a synthetic cry3a gene under the control of CaMV35S promoter as well as the nptII gene as selectable marker. Transformed explants were cultured on callus-induction medium, and the germinated somatic embryos were then transferred to the regeneration medium. The primary transformants were evaluated by PCR and Southern blot analysis. The results indicated successful integration of the target gene into the genomes of primary transgenic lines. Moreover, the expression of Cry3a protein in the transgenic plants was confirmed by ELISA method. Three transgenic lines, including TL6, TL8 and TL11 showed significantly higher levels of insect resistance against H. postica larvae (mortality rate of 73–90 % after infestation), in comparison with the control plants during the two-year bioassays. All transgenic plants were fertile and no irregular behavior in terms of growth and the morphological traits were observed. Transgenic plants developed during the course of this study are currently being grown in greenhouse and will be crossed with each other for seed production.

Keywords

Agrobacterium tumefaciens Alfalfa weevil cry3a Hypera postica Transgenic alfalfa 

Abbreviations

Bio2Y

Blaydes 1966 medium

CaMV

Cauliflower mosaic virus

DIG

Digoxigenin-dUTP

2,4-D

2,4-dichlorophenoxyacetic acid

ELISA

Enzyme-linked immunosorbent assay

MS

Murashige and Shoog medium (1962)

nptII

Neomycin phosphotransferase gene

Nos

Nopaline synthase

OD

Optical density

Notes

Acknowledgments

This work was supported by a grant received from the Agricultural Research and Education Organization of Iran (AREO). We wish to thank all our colleagues in the Department of Plant Tissue Culture and Genetic engineering of ABRII for their support and technical assistance. The authors would also like to extend their appreciation to Assist. Prof. Meisam Tabatabaei for his critical review of the manuscript in terms of its academic English writing.

References

  1. Austin S, Bingham ET, Mathews DE, Shahan MN, Will J, Burgess RR, Cassells AC, Jones PW (1995) Production and field performance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase. Euphytica 85:381–393CrossRefGoogle Scholar
  2. Bao A-K, Wang SM, Wu GQ, Xi JJ, Zhang JL, Wang CM (2009) Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.). Plant Sci 176:232–240CrossRefGoogle Scholar
  3. Bharathi Y, Vijayakumar S, China PI, Dasavantha R, Venkateswara R (2008) Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests. BMC Plant Biol 8:102–117CrossRefGoogle Scholar
  4. Bhat SR, Srinivasan S (2002) Molecular and genetic analyses of transgenic plants considerations and approach. Plant Sci 163:673–681CrossRefGoogle Scholar
  5. Bingham E (1991) Registration of alfalfa hybrid Regen-SY germplasm for tissue culture and transformation. Crop Sci 31:1098–1118CrossRefGoogle Scholar
  6. Blaydes DF (1966) Interaction of kinetin and various inhibitors in the growth of soybean tissue. Physiol Plant 19:748–753CrossRefGoogle Scholar
  7. Chandra A, Pandey KC (2008) Effect of proteinase inhibitors on Indian alfalfa weevil (Hypera postica Gyll.) growth and development. Acta Physiol Plantarum 30:501–505CrossRefGoogle Scholar
  8. Desgagnés R, Serge L, Guy A, Habib YC, Jacques L, Réal M, Louis-P V (1995) Genetic transformation of commercial breeding lines of alfalfa (Medicago sativa). Plant Cell Tiss Org Cult 42:129–140CrossRefGoogle Scholar
  9. Ghareyazie B, Alinia F, Menguito CA, Rubia LG, Palma JM, Liwanag EA, Cohen MB, Bennett J, Khush GS (1997) Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA(b) gene. Mol Breed 3:401–414CrossRefGoogle Scholar
  10. González et al (2008) Efficient regeneration and Agrobacterium tumefaciens mediated transformation of recalcitrant sweet potato (Ipomoea batatas L.) cultivars. Asia Pac J Mol Biol Biotechnol 16:25–33Google Scholar
  11. Hibbard BE, Clark TL, Ellersieck MR, Meihls LN, El Khishen AA, Kaster V, Steiner HY, Kurtz R (2010) Mortality of western corn rootworm larvae on MIR604 transgenic maize roots: field survivorship has no significant impact on survivorship of F1 progeny on MIR604. J Econ Entomol 103:2187–2196PubMedCrossRefGoogle Scholar
  12. Hoefte H, Whiteley HR (1989) Insecticidal Crystal proteins of Bacillus thuringiensisa. Microbiol Rev 53:242–255Google Scholar
  13. James C (2011) Global status of commercialized biotech/GM crops: brief 43, International Service for the Acquisition of Agribiotech Applications ISAAA, pp 1-16Google Scholar
  14. Jiang Q, Zhang JY, Guo X, Monteros MJ, Wang ZY (2009) Physiological characterization of transgenic Alfalfa (Medicago sativa) plants for improved drought tolerance. Int J Plant Sci 170:969–978CrossRefGoogle Scholar
  15. Jin T, Chang Q, Li W, Yin D, Li Z, Wang D, Liu B, Liu L (2010) Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa. Plant Cell Tissue Organ Cult 100:219–227CrossRefGoogle Scholar
  16. Jouzani GS, Komakhin RA, Piruzian ES (2005) Comparative study of the expression of the native, modified, and hybrid cry3a genes of Bacillus thuringiensis in prokaryotic and eukaryotic cells. Russ J Genet 4:116–121CrossRefGoogle Scholar
  17. Jouzani GS, Goldenkova IV, Piruzian ES (2008) Expression of hybrid cry3aM-licBM2 genes in transgenic potatoes (Solanum tuberusom). Plant Cell Tissue Organ Cult 92:321–325CrossRefGoogle Scholar
  18. Kechang L, Ping Z, Cash D (2009) Biology and management of major alfalfa diseases and pests. In: Cash D, Yuegao H, Kechang L, Suqin W, Ping Z, Rong G (eds) Alfalfa management guide for ningxia. China Agricultural Press, China, pp 37–62Google Scholar
  19. Khanjani M (ed) (2005) Alfalfa pests. In: Crop pests in Iran. Ali Sina University Press, Iran, pp 121-138Google Scholar
  20. Krieg, Aloisius, Huger, Alois S (1989) Protein toxin from Bacillus thuringiensis which is toxic to coleopteran. Patent US 4889918Google Scholar
  21. Kuiper HA, Kleter GA, Hub N, Kok EJ (2001) Assessment of the food safety issues related to genetically modified foods. Plant J 27:503–528PubMedCrossRefGoogle Scholar
  22. Li H, Jinhua L, Hemphill JK, Wang JT, Gould J (2001) A rapid and high yielding DNA miniprep for cotton (Gossypium spp.). Plant Mol Biol Rep 19:183a–183eGoogle Scholar
  23. Ma XF, Tudor S, Butler T, Ge Y, Xi Y, Bouton J, Harrison MYuZW (2012) Transgenic expression of phytase and acid phosphatase genes in alfalfa (Medicago sativa) leads to improved phosphate uptake in natural soils. Mol Breed 30:377–391PubMedCrossRefGoogle Scholar
  24. Mesfin TM, Matthew DD, Deborah A, Samac C, Vance P (2005) Transgenic alfalfa secretes a fungal endochitinase protein to the rhizosphere. Plant Soil 269:233–243CrossRefGoogle Scholar
  25. Meyer P (1985) Understanding and controlling transgene expression. Trends Biotechnol 13:332–337CrossRefGoogle Scholar
  26. Pires AS, Rosa S, Castanheira S, Fevereiro P, Abranches R (2012) Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago. Plant Cell Tissue Organ Cult 110:171–181CrossRefGoogle Scholar
  27. Samac DA (1995) Strain specificity in the transformation of alfalfa (Medicago sativa) by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 43:271–277Google Scholar
  28. Samac DA, Smigocki AC (2003) Expression of oryzacystatin I and II in transgenic alfalfa increases resistance to the root-lesion nematode. Phytopathol 93:799–804CrossRefGoogle Scholar
  29. Schluter U, Benchabane M, Lie AM, Kiggundu A, Vorster J, Goulet MC, Cloutier C, Michaud D (2010) Recombinant protease inhibitors for herbivore pest control: a multitrophic perspective. J Exp Bot 61:4169–4183PubMedCrossRefGoogle Scholar
  30. Smith DS, Maxwell PW, De Boer SH (2004) Method for the detection of synthetic cry3A in transgenic potatoes. J Agric Food Chem 25:809–815CrossRefGoogle Scholar
  31. Srivastava V, Ow DW (2001) Single-copy primary transformants of maize obtained through the co-introduction of a recombinase-expressing construct. Plant Mol Biol 46:561–566PubMedCrossRefGoogle Scholar
  32. Tesfaye M, Samac DA, Lamb JFS (2009) Alfalfa. In: Chittaranjan K, Timotry CH (eds) Compendium of transgenic crop. Wiley, New York, pp 199–210Google Scholar
  33. Tohidfar M, Ghareyazie B, Mousavi M, Yazdani S, Golabchian R (2008) Agrobacterium mediated transformation of cotton (Gossypium hirsutum) using a syntheticcry1Ab gene for enhanced resistance against Heliothis armigera. Iran J Biotechnol 6:164–173Google Scholar
  34. Wilhite SE, Elden TC, Brzin J, Smigocki AC (2000) Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors. Insect Biochem Mol Biol 30:1181–1188PubMedCrossRefGoogle Scholar
  35. Yan LP et al (2012) Physiological responses to salt stress of T2 alfalfa progenies carrying a transgene for betaine aldehyde dehydrogenase. Plant Cell Tissue Org Cult 108:191–199CrossRefGoogle Scholar
  36. Zhang Y, Liu J (2011) Transgenic alfalfa plants co-expressing glutathione S-transferase (GST) and human CYP2E1 show enhanced resistance to mixed contaminates of heavy metals and organic pollutants. J Hazard Mat 189:357–362CrossRefGoogle Scholar
  37. Zhang B, Chen M, Zhang X, Luan H, Diao S, Tian Y, Su X (2011) Laboratory and field evaluation of the transgenic Populus alba × Populus glandulosa expressing double coleopteran-resistance genes. Tree Physiol 31:567–573PubMedCrossRefGoogle Scholar
  38. Zhang YM, Liu ZH, Wen ZY, Zhang HM, Yang F, Guo XL (2012) The vacuolar Na+–H+ antiport gene TaNHX2 confers salt tolerance on transgenic alfalfa (Medicago sativa). Funct Plant Biol, Published online 18 July 2012Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Masoud Tohidfar
    • 1
    Email author
  • Naser Zare
    • 1
  • Gholamreza Salehi Jouzani
    • 2
  • Seide Maryam Eftekhari
    • 1
  1. 1.Department of Plant Tissue Culture and Gene TransformationAgricultural Biotechnology Research Institute of Iran (ABRII)KarajIran
  2. 2.Microbial Biotechnology and Biosafety DepartmentAgricultural Biotechnology Research Institute of Iran (ABRII)KarajIran

Personalised recommendations