Increasing productivity of guar and guar gum

1. Increasing productivity of guar and guar gum G. S. Randhawa Department of Biotechnology IIT Roorkee

2. GUAR (CLUSTER BEAN) A NEGLECTED MIRACLE BEAN

3. The economic times “Guar gum has overtook traditional heavyweights of India’s top farm export; rice and cotton, and seems to make its place into the league of top ten shipments of the country” http://articles.economictimes.indiatimes.com/keyword/guar-gum (March 2013)

4. A strong need to enhance the productivity of guar crop and guar gum BUT HOW ???

5. Approaches to enhance the productivity • Increasing area under cultivation • Improving agronomic practices • Controlling pests and diseases • By developing varieties having the following characterstics: • High yield • Stress tolerance • Disease and pest resistance • Improved gum quantity and quality

6. PROBLEMS IN ENHANCING PRODUCTIVITY • Poor research funding • Less number of scientists • Poor infrastructure facilities

7. Research approaches for improvement • Classical / traditional approaches • Biotechnological approaches

8. Classical/ traditional approaches • Germplasm collection • Screening of germplasm • Hybridization and selection

9. BIOTECHNOLOGICAL APPROACHES • Molecular markers • Identification of genes • Characterization of guar proteins • Regulation of gene expression • Genetic manipulation • Cell and tissue culture • Sequencing of guar genome

10. Molecular MARKERS A fragment of DNA that is associated with a certain location within the genome is called molecular marker Various types of molecular markers: RFLP, RAPD, AFLP, ISSR, SSR, SNP Studies in guar: RAPD (Liu et al., 2009, Punia et al., 2009 and Nagesh et al., 2013) ISSR (Nagesh et al., 2013) SSR (Nagesh et al., 2014)

11. Molecular characterization of cluster bean (Cyamopsis tetragonoloba L. Taub) using RAPD and ISSR markers Nagesh et al., 2013

12. Identification and Characterization of EST-SSR Markers • A total of 907 SSR-containing sequences were identified • Out of 224 primer pairs, 3 resulted in the amplification of SSR-containing sequences producing reliable and reproducible DNA bands in Cyamopsis tetragonoloba, C. serrata, and C. senegalensis, respectively Banding pattrens obtained from amplification of SSR markers Nagesh et al., 2014

13. sucrose fructose-6-phosphate phosphomannoisomerase (PMI) hexokinase UDP fructose mannose-6-phosphate mutase mannose-1-phosphate GTP GDP-mannose pyrophosphorylase epimerase PPase 2Pi PPi GDP-mannose UDP-galactose b-mannan synthase UDP gal b-mannan -man-man-man-man- -man-man-man-man- a-galactosyltransferase galactomannan gal KSD/020503 Pathway to galactomannan formation in plants UDP-glucose

14. IDENTIFICATION OF GENES Genes involved in gum synthesis Mannan synthase (Dhugga et al., 2004) Galactomannan galactosyltransferase (Edwards et al., 1999) α-galactosidase (Reid and Meier, 1973) Genes responsible for stress tolerance ????? Genes responsible for plant growth and development ?????

15. ß-MANNAN SYNTHASE The first plant cell wall hemicellulosic gene cloned Dr. Kanwarpal Singh Dhugga who cloned ManS gene of guar Dhugga et al., Science, 2004

16. Pioneer Hi-Bred International Inc. where ManS gene of guar was cloned

17. In our lab cloning, expression, purification and characterization of guar plant proteins is in progress Peptide mass fingerprinting of a guar protein (Unpublished data from our lab)

18. Genes responsible for stress tolerance in guar Studying of the pathway responsible for stress tolerance in guar using the physiological, biochemical and molecular approachesis being carried out in our lab. Identification of genes responsible for stress tolerance is in progress. (Unpublished data)

19. Potential strength of promoters REGULATION OF GENE EXPRESSION Characterization of mannan synthase promoter from guar Analysis of GUS expression in different tissues of transgenic alfalfa plants carrying GUS reporter genes under control of the MS, Phas and 35S promoters. Promoter activity was evaluated in roots, leaf, flowers, pods, seed coat, endosperm and embryos, respectively: a, d, g, j, m, p, s 35S promoter b, e, h, k, n, q, t MS promoter c, f, i, l, o, r, u b-phaseolin promoter Naoumkina and Dixon, 2011

20. GENETICALLY MODIFIED PLANTS In vivo modification of cell wall polysaccharide galactomannan of guar transformed with alpha-galactosidase gene cloned from senna Sequence of Senna alpha galactosidase and Restriction enzyme map of the plant transformation vector, pBS-GNG-GAL, used to transform guar. Joersbo et al. 2001

21. In our lab work on RNAi in guar is in progress Transformed guar plant showing expression of reporter gus gene in leaf tissue (Unpublished data)

22. Genetically Modified Guar Plantlets (Unpublished data)

23. PLANT CELL AND TISSUE CULTURE • Explants used: • Cotyledon • Hypocotyl • Cotyledonary node • (from in vitro germinated seedlings) • Different combinations of plant growth hormones used: • For regeneration: • Different concentrations of NAA -BAP, NAA-KN, IAA-KN, IAA-BAP, 2,4-D-BAP and • 2,4-D-KN combinations • For elongation of regenerated shoots: • Different concentrations of BA-IAA-GA3 in combination • For rooting: • IAA and IBA alone

24. Verma et al., 2013

25. In vitro regeneration of complete plantlets in a single culture in guar Verma et al., 2013

26. Our Team Kulvinder S. Gill Kanwarpal S. Dhugga Pranita Bhatele Nagesh K A Swati Verma Manisha Choudhary Shilpi Kumari Umesh K. Tanwar Shalini Pareek Pallavi Gahlot Prof. S.K. Tripathi Prof. S.M. Sondhi Prof. Pravindra Kumar Navneet Kaur Deepa Dewan Nishu Mittal Poonam Jaiswal

27. Sardar Tara Singh JiMy Mentor

28. Thank you

29. My website WWW.GSRANDHAWA.IN My email SHARNFBS@IITR.AC.IN