Optimization of genetic transformation in Lentil (Lens culinaris Medik.) using Agrobacterium tumefaciens

Document Type : Original Articles

Authors

• Fatemeh zaker tavallaie ¹
• Abdorreza Bagheri ²
• Behzad Ghareyazi ³
• Kiran Komar Sharma ⁴

¹ Complex Higher education of shirvan

² Ferdowsi University of Mashhad

³ Agricultural Biotechnology Research Institute of Iran (ABRII),

⁴ International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)

10.22067/ijpr.v8i2.52960

Abstract

Lentil (Lens culinaris Medik.) may have been one of the first agricultural crops grown more than 8,500 years ago and belongs to genus Lens from family leguminous and it is diploid (2n=14). Lentil is a seed propagating, self-pollinating crop originating from Near East. Lentil contains high percentage of protein, and has high nutrient value and easy digestibility. Genetic engineering has high potential to improve tolerance of lentil against biotic and abiotic stresses. Genetic engineering may also have a role in eliminating anti-nutritional factors and improving the nutritional quality of lentil proteins. Lentil can also be employed as host to produce drug using gene transformation. To date only a few reports are available on attempts to transform lentil. In this research we attempted to optimize conditions of gene transformation in lentil.
Materials & Methods
Seeds of lentil (Lens Culinaris Medik) variety of Gachsaran (ILL6212) were used in present investigation, collected from Shirvan Agriculture Research Center, northeast of Iran. The Explants of Cotyledon with slight part of Embryo Axes (CEA) were prepared as described in Zakertavallaie et al, (2011) study. The explants were kept on Shoot Induction Media (SIM) including MS media supplemented with 7.5µM 2ip, 4 µM Kin and 2 µM TDZ for 8 days. Then explants containing shoots sub cultured on MS media for another 7 days. 15 days explants containing shoots were used for co-cultivation with Agrobacterium. Agrobacterium tumifaciens strain C58 containing binary vector of pCAMBIA1301 was used for transformation experiments. This plasmid contains a reporter gene gus and a selectable gene hptΙΙ, into
T-DNA for selection of putative transgenic shoots and selectable marker gene nptΙΙ to select transformed bacteria in backbone sequence. Suspension of agrobacterium in 1/2 MS with 4 concentration of OD: 0.13, OD: 0.18, OD: 0.22 and OD: 0.26 was compared to use in co-cultivation media. Explants containing shoots were used for co-cultivation. There was compared two type of preparation of explants to use in co-cultivation. About some of explants, only tip of the shoots removed using scalpel but about some of them almost of shoots removed. All of explants were submerged in Agrobacterium suspension for 2 second and were co-cultured on shoot induction media. Cu-cultivated explants containing shoots were cultured on MS media containing 250mg/lit cefotaxim for 7 days. In this stage shoots transferred to elongation media including MS supplemented with 4µM 2ip and 1µM Kin containing 3 mg/L hygromycine to select transgenic shoots and 200 mg/lit cefotaxim to kill bacteria. The hygromycine was increased with each subculture at 7 days intervals up to 5 mg/lit and the non-health and non-green shoots were discarded in each subculture. Elongation of shoots, rooting, hardening and glasshouse growth was done according to Zakertavallaie et al, (2011) protocol. Gus assay carried out before root induction using Hay et al. (2004) protocol and Blue dot were observed under microscope. DNA was extracted from leaves of both non transgenic and putative transgenic plants using modified CTAB protocol. DNA was subjected to Polymerase change reaction using the hyg and gus primers with. PCR was carried out with following condition: PCR reaction mix of 25 µl contained 2 µl genomic DNA (100ng/µl), 2.5µl 10X buffer, 2 µl MgCl2 (50mM), 0.5 µl dNTP (10Mm) and 0.5 µl Taq DNA polymerase (5u/µl). The concentration of primers were also 100 pmol/ µl. in PCR amplification DNA denatured at 94ºC for 5 min then followed by 35 cycles with 1 min at 94ºC, I min at annealing temperature (for gus gene at 58 ºC and for hpt gene at 59 ºC), 1 min at 72 ºC. final extension was carried out at 72 ºC for 10 min. PCR product was run on 1.2% agarose gel and observed under UV after staining with ethidium bromide.
Results & Discussion
Explants after shoot induction were prepared for co-cultivation. Co-cultivation period was 72 hours. Concentration of Agrobacterium was important. Acetoceringone has no effect on success of gene transformation. Concentrations of 3 to 5 mg/lit hygromycine were suitable for selection media in shooting stage. Gus assay confirmed gene transformation in putative transgenic shoots. Also PCR reaction confirmed existence of gus and hpt genes in plants.
Conclusion
The employed protocol in this research can be used for gene transformation of lentil using another gene to improve it about other treats such as enhancement of tolerance to biotic and abiotic stresses.

Keywords

• Genetic engineering
• Gus gene
• Lentil (Lens culinaris Medik.)
• Regeneration
• Transgenic plant

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References

1. Akcay, U.C., Mahmoudian, M., Kamci, H., Yucel, M., and Oktem, H.A. 2009. Agrobacterium tumefaciens-mediated genetic transformation of a recalcitrant grain legume, lentil (Lens culinaris Medik). Plant Cell Report 28: 407-417.

2. Bohra, A., Pandey, M.K., Jha, U.C., Singh, B., Singh, I.P., Datta, D., Chaturvedi, S.K., Nadarajan, N., and Varshney, R.K. 2014. Genomics-assisted breeding in four major pulse crops of developing countries: present status and prospects. Theoretical and Applied Genetics 127: 1263-1291.

3. Chowrira, G.M., Akella, V., and Lurquin, P.F. 1995. Electroporation-mediated gene transfer into intact nodal meristems in planta. Generating transgenic plants without in vitro tissue culture. Molecular Biotechnology 3: 17-23.

4. Gulati, A., Schryer, P., and McHughen, A. 2001. Regeneration and micrografting of lentil shoots. In vitro Cellular and Development Biology 37: 798-802.

5. Gulati, A. , Schryer, P., and Mchughen, A. 2002. Production of fertile transgenic lentil (Lens culinaris Medik.) plants using particle bombardment. In vitro Cellular and Development Biology-Plant 38: 316-324.

6. Hay, I., Lachance, D.A., Von Aderkas, P., and Charest, P.J. 1994. Ransient chimeric gene expression in pollen of five conifer species following microparticle bombardment. Canadian Journal of Forest Research 24(12): 2417-2423.

7. Khatib, F., Makris, A., Yamaguchi-Shinozaki, K., Kumar, S., Sarker, A., Eroskine, W., and Baum, M. 2011. Expression of the DREB1A gene in lentil (Lens culinaris Medik. subspculinaris) transformed with the Agrobacterium system.Crop Pasture Science 62: 488-495.

8. Kumar, S.K., Barpete, S., Kumar, J., Gupta, P., and Sarker, A. 2013. Global lentil production: constraints and strategies. SATSAMukhapatra. Annual Review of Information Science and Technology 17: 1-13.

9. Maccarrone, M., Veldink, GA., Finazzi, A.A., and Vliegenthart, J.F. 1995. Lentil root protoplasts: a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules. Biochimicaet Biophysica Acta 1243: 136-142.

10. Mahmoudian, M., Yucel, M., and Oktem, H.A. 2002. Transformation of lentil (Lens culinaris Medik.) cotyledonary nodes by vacuum infiltration of Agrobacterium tumefaciens. Plant Molecular Biology Reports 20: 251-257.

11. Öktem, H.A., Mahmoudian, M., Eyidoðan, F., and Yücel, M. 1999. Gus gene delivery and expression in lentil cotyledonary nodes using particle bombardment. Lens Newsletter 26: 3-6.

12. Porebski S., Bailey L.G., and Baum, B.R. 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 15: 8-15.

13. Rajendran, S.K., Kumar, J., Hamwieh, A., and Baum, M. 2015. Current knowledge in lentil genomics and its application for crop improvement. Front Plant Science 6: 78-90.

14. Sarker, R.H. ,Biswas, A. , Mustafa, B.M. ,Mahbub, S., and Hoque, M.I. 2003. Agrobacterium-mediated transformation of lentil. Plant Tissue Culture 13: 1-12.

15. Sheikholeslam, S.N., and Weeks, D.P. 1987. Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Molecular Biology 8: 291-308.

16. Warkentin, T.D., and McHughen, A. 1993. Regeneration from lentil cotyledonary nodes and potential of this explant for transformation by Agrobacterium tumefaciens. Lens Newsletter 20: 26-28.

17. Zaker Tavallaie, F., Ghareyazie, B., Bagheri, A., and Sharma, K.K. 2011. Lentil regeneration from cotyledone explant bearing a small part of the embryo axis. Plant Tissue Culture and Biotechnology 21: 169-180.