Genetic Transformation and Functional Genetics Lab

Tools of genetic transformation are increasingly contributing to a better understanding of the genetic basis of plant growth and development in addition to development of novel varieties. At IFGTB, a breeding-integrated transgenic programme is ongoing for development of varieties and clones with increased salt and pest tolerance. Development of methods for regeneration and transformation of tree species are challenging and significant advances have been made in Eucalyptus camaldulensis. Salt tolerant tree species like Casuarinas are being prospected for major genes conferring salt tolerance for use in marker assisted selection and transgenic programmes. Projects on using gene silencing techniques for functional analysis of these genes are ongoing. Initiatives are underway in using RNAi for incorporation of resistance against the Eucalyptus pest, Leptocybe invasa. Studies have also been initiated for gene-silencing based control of teak insect pest Hyblaea puera. As these take shape, initiatives for engineering other traits like improved productivity under drought conditions, and improved pulping traits will also be taken up through national and international collaboration, in the process, making IFGTB an international centre for transgenic studies in trees. Presently, the laboratory is a partner of the multi-national network on "Transcriptome analysis of Salt tolerance in Casuarina trees" by the Joint Genome Institute, US. The lab has attracted CSIR scholars for Ph.D. studies and has hosted a Senegalese post-doctoral researcher through C.V.Raman fellowship. The laboratory houses facilities like the Gene gun, Electroporator, Sonicator, Stereo-fluorescent microscope, PCR and Real-time PCR. The Functional Genetics Experimental Facility enables conduct of transgenic research under contained conditions as per the national biosafety regulations. The projects have enabled the conduct of a 2 weeks training program on Genetic Engineering to ICFRE scientists.

Vision

To reach out to farmers through development of transgenic trees with enhanced productivity and quality traits for growing in areas affected by biotic and abiotic stresses.

Mission    

To evolve transgenic approaches in forestry species for functional characterization of genes and genetic modification of desired traits.

Focus species:             Casuarina sp., Eucalyptus sp.,

Other species :           Prosopis juliflora, Acacia nilotica, Pongamia pinnata, Kandelia candel, Bruguiera gymnorrhiza, Bruguiera sexangula, Bruguiera cylindrica

Achievements

Ongoing Projects/ Studies

  • • Development of methods for functional analysis of genes involved in salt tolerance in Eucalyptus tereticornis. ICFRE: 30.93 lakhs (2009-14).
  • • Determination of target genes in Leptocybe invasa for engineering resistance in Eucalyptus through gene- silencing approaches. ICFRE: 23.50 lakhs (2010-2014)
  • • Incorporating resistance in Eucalyptus to Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae) through expression of insect specific dsRNA. ICFRE: 29.754 lakhs (2012-2016).
  • • Identification and functional analysis of genes of the teak insect pest Hyblaea puera Cramer (Hyblaedae: Lepidoptera) for the development of gene silencing based pest control. CSIR PhD student ( Mrs. Sowmiya Rani ) study (2010-2014)

Completed Projects

  • • Web enabled database and analysis of gene sequences implicated in abiotic stress tolerance for screening gene homologues in salt tolerant tree species. DBT: 20.096 lakhs (2009-2012)

International Collaboration

Partner of the international network on "Transcriptome analysis of Salt tolerance in Casuarina trees" by the Joint Genome Institute, US jointly proposed by

Dr. Laurent Laplaze, Research Director, IRD, Dakar, Senegal.

Dr. Louis Stephen TISA, Professor of Microbiology and Genetics, University of New Hampshire, USA.

Dr. Valérie Hocher,Research Scientist, IRD, Montpellier, France

Dr. Diegane Diouf, Associate Professor, Université Chiekh Anta Diop, Dakar (UCAD), Senegal

Dr. Mathish Nambiar-Veetil, Scientist D, Institute of Forest Genetics and Tree Breeding, Coimbatore, India

A post-doctoral researcher, Dr. Nathalie Diagne, from UCAD, Senegal (one of the four international partner institutes on the JGI project on Transcriptome analysis of salt tolerance in Casuarina trees) was hosted for a six months CV Raman Fellowship

With IRD, Montpellier, France

A study on "Development of post-transcriptional gene silencing approaches as a tool for the functional analysis of symbiotic genes in the tropical actinorhizal tree Casuarina glauca" was carried out by Dr. Mathish N.V at Dr. Didier Bogusz's Rhizogenesis Lab at IRD, France with the guidance of Dr. Claudine Franche, Member, High Council of Biotechnology, and Dr. Hassen Gherbi, Research Director.

Inter-divisional collaboration

Shri. R. Vivekanandan, Scientist E, Computer Division

Dr. Jacob, J. P. Scientist E, Forest Protection Division

Dr. V. Sivakumar, Scientist E, Genetics and Tree Breeding Division

Dr. Kannan C.S. Warrier, Scientist D, Genetics and Tree Breeding Division

Dr. C. Buvaneswaran, Scientist E, Forestry, Land use and Climate Change Division

Lab Members

Dr. Mathish Nambiar-Veetil, Scientist E

Mr. Balasubramanian, A, RA-I

Mrs. Sudha, S, TA-I

Mrs. Sowmiya Rani, K, CSIR-SRF

Mr. Rathish Prabhakaran, SRF

Ms. Vikashini, B, JRF

Mr. Vijaya kumar, S, Project Assistant

Previous lab members

Mr. Selvakesavan, R.K, SRF (Presently at Portugal); Mr. Venkatachalam,R, SRF (Presently at ITC); Mrs. Tressa Hamalton, Project Assistant (Presently Scientist at TFRI, Jabalpur); Mrs. Thushara Aravindh, CSIR-JRF; Ms. Sanu Mary Abraham, Project Assistant;Mr. Balaji,S, Project Assistant (Presently at CPMB, TNAU); Ms. Yazhini.P, Project Assistant (Presently at IFGTB); Ms. Dhanya Nair, Project Assistant; Mr. Niranjan. P, Project Assistant (Presently at U.S); Ms. Sangeetha, M, JRF (Presently at Sankara Nethralaya); Mr.Vignesh,M, JRF (Presently at Parry’s sugar); Mr. Aravinthakumar, V, Project Assistant (Presently at Monsanto); Mr. Naveen kumar, A, Project Assistant (Presently at CPMB, TNAU); Ms. Lalitha, S, JRF (Presently at RGCB)

Facilities

Stereo fluorescent microscope

Electroporator

Gene gun

Transgenic green house (Functional Genetics Experimental Facility)

Journal Publications (since 2006)

Svistoonoff, S., Benabdoun, F.M., Mathish, N.V., Imanishi, L., Vaissayre, V., et al. 2013 The Independent acquisition of plant root nitrogen-fixing symbiosis in
      Fabids recruited the same genetic pathway for nodule organogenesis. Public Library of Science ONE 8(5): e64515. doi:10.1371/journal.pone.0064515

Benabdoun, F.M., Mathish, N.V., Imanishi, L., Svistoonoff, S., Ykhlef, N., Gherbi, H., and Franche, C. 2011. Composite actinorhizal plants with transgenic roots for
       the study of symbiotic associations with Frankia. Journal of Botany, 2011, doi:10.1155/2011/702947.

Balasubramanian, A., Venkatachalam, R., Selvakesavan, R.K., Sanu Mary, A., Gherbi, H., Svistoonoff, S., Franche, C., Bogusz, D., Krishna Kumar, N., and
       Mathish, N.V. 2011. Optimisation of methods for Agrobacterium rhizogenes mediated generation of composite plants in Eucalyptus camaldulensis. BioMed
       Central proceedings. 5: 45.

Mathish, N.V., Sangeetha, M., Sowmiya Rani, K.S., Aravinthakumar, V., Selvakesavan, R.K., Balasubramanian, A., Venkatachalam, R., Sanu Mary, A., Jacob,
       J.P., and Krishna Kumar, N. 2011. Identification of insect-specific target genes for development of RNAi based control of the Eucalyptus gall pest Leptocybe
       invasa Fisher & La Salle (Hymenoptera: Eulophidae). BioMed Central proceedings. 5: 98.

Perrine-Walker, F., Gherbi, H., Imanishi, L., Hocher, V., Ghodhbane-Gtari, F., Lavenus, J., Benabdoun, M., Mathish, N.V., Svistoonoff S., Laplaze L. 2011.
         Symbiotic signaling in actinorhizal symbioses. Current Protein and Peptide Science. 12: 156-164.

Svistoonoff, S., Gherbi, H., Mathish, N. V., Zhong, C., Michalak, Z., Laplaze, L., Vayssaire, V., Auguy, F., Hocher, V., Doumas, P., Bonneau, J., Bogusz, D., and
       Franche, C. 2009. Contribution of transgenic Casuarinaceae to knowledge of the actinorhizal symbiosis. Symbiosis. 50: 3-11.

Gherbi, H., Mathish, N. V., Zhong,C., Félix, J., Autran, D., Girardin, R., Vaissayre, V., Auguy, F., Bogusz, D., and Franche, C. 2008. Post- transcriptional gene
       silencing in the root system of the actinorhizal tree Allocasuarina verticillata. Molecular Plant Microbe Interactions. 21: 518–524.

Tripathi, S.B., Mathish, N.V., Gurumurthi, K. 2006. Use of genetic markers in the management of micropropagated Eucalyptus germplasm. New Forests. 31:361-
       372

GenBank Submissions: Abiotic stress tolerance

S. No
Plant Species
Gene
Source
Accession No
bp
Gene function
1.
Casuarina equisetifolia
NHX1 gene, partial cds
Genomic DNA
JN629033
330
Na+/H+ antiporter
2.
AKT1 gene, partial cds
Genomic DNA
JX840853
236
K+ channel
3.
Actin mRNA, partial cds
RNA/cDNA
JQ837965
204
Housekeeping gene
4.
Eucalyptus camaldulensis
NHX1 gene, partial cds
Genomic DNA
JN157814.1
494
Na+/H+ antiporter
5.
AKT1 gene, partial cds
Genomic DNA
JX840853
236
K+ channel
6.
Actin gene, partial cds
Genomic DNA
JN157813.1
311
Housekeeping gene
7.
Eucalyptus tereticornis
HKT1 gene, partial cds
Genomic DNA
JF86711
638
Na+/K+ symporter
8.
NHX1 gene, partial cds
Genomic DNA
JN157810.1
614
Na+/H+ antiporter
9.
Acacia nilotica
AKT1 gene, partial cds
Genomic DNA
JX840860
261
K+ channel
10.
NHX1 mRNA, partial cds
RNA/cDNA
JQ837963
348
Na+/H+ antiporter
11.
Actin mRNA, partial cds
RNA/cDNA
JN157811.1
201
Housekeeping gene
12.
Millettia pinnata (Pongamia pinnata)
NHX1 gene, partial cds
Genomic DNA
JX218027.1
385
Na+/H+ antiporter
13.
Actin mRNA, partial cds
RNA/cDNA
JN157812.1
213
Housekeeping gene
14.
Prosopis juliflora
HKT1 mRNA, partial cds
RNA/cDNA
JX679724
219
Na+/K+ symporter
15.
AKT1 gene, partial cds
Genomic DNA
JX840855
300
K+ channel
16.
AKT1 mRNA, partial cds
RNA/cDNA
JX840859
291
K+ channel
17.
NHX1 gene, partial cds
Genomic DNA
JN629034
371
Na+/H+ antiporter
18.
Kandelia candel
NHX1 gene, partial cds
Genomic DNA
JQ837964
725
Na+/H+ antiporter
19.
AKT1 gene, partial cds
Genomic DNA
JX840858
273
K+ channel
20.
Actin gene, partial cds
Genomic DNA
JX679723
204
Housekeeping gene
21.
Bruguiera gymnorrhiza
NHX1 gene, partial cds
Genomic DNA
JX679717
355
Na+/H+ antiporter
22.
Actin gene, partial cds
Genomic DNA
JX679721
209
Housekeeping gene
23.
Bruguiera sexangula
NHX1 gene, partial cds
Genomic DNA
JX679719
351
Na+/H+ antiporter
24.
AKT1 gene, partial cds
Genomic DNA
JX840856
357
K+ channel
25.
Actin gene, partial cds
Genomic DNA
JX679722
221
Housekeeping gene
26.
Bruguiera cylindrica
NHX1 gene, partial cds
Genomic DNA
JX679718
445
Na+/H+ antiporter
27.
AKT1 gene, partial cds
Genomic DNA
JX840857
409
K+ channel
28.
Actin gene, partial cds
Genomic DNA
JX679720
203
Housekeeping gene

 

GenBank Submissions: Insect growth and development

S. No
Insect Species
Type
Gene
Source
Accession No
bp
1.
Leptocybe  invasa
Target genes
Chitin synthase gene, partial cds
Genomic DNA
JF772551.1
624
2.
Chitinase mRNA, partial cds
RNA/cDNA
KC818286.1
596
3.
Housekeeping genes
Elongation factor 1- alpha mRNA, partial cds
RNA/cDNA
JN381022.1
587
4.
Elongation factor 1- alpha gene, partial cds
Genomic DNA
JF772552.1
534
5.
Beta- tubulin mRNA partial cds
RNA/cDNA
KC818287.1
691
6.
Actin gene, partial cds
Genomic DNA
JN673812.1
189
7.
Hyblaea puera
Target genes
Chitin synthase gene, partial cds
Genomic DNA
JF774409.1
1,032
8.
Chitin synthase mRNA, partial cds
RNA/cDNA
JQ289043.1
468
9.
Chitin synthase gene  clone 1, partial cds
Genomic DNA
KC121027.1
204
10.
Chitin synthase gene clone 2, partial cds
Genomic DNA
KC121028.1
741
11.
Chitinase gene, partial cds
Genomic DNA
JX101956.1
480
12.
Ecdysone receptor gene, partial cds
Genomic DNA
JX644041.1
751
13.
SXR-like protein mRNA
RNA/cDNA
JQ319890.1
630
14.
Housekeeping genes
Actin gene, partial cds
Genomic DNA
JN368462.1
619
15.
Beta-tubulin mRNA, partial cds
RNA/cDNA
KC880336.1
560