Increasing productivity of guar and guar

gum

G. S. RandhawaDepartment of Biotechnology

IIT Roorkee

G. S. RandhawaDepartment of Biotechnology

IIT Roorkee

GUAR (CLUSTER BEAN)

A NEGLECTED MIRACLE BEAN

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)

A strong need to enhance the productivity of guar crop and guar gum

BUT HOW ???

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

PROBLEMS IN ENHANCING PRODUCTIVITY

• Poor research funding

• Less number of scientists

• Poor infrastructure facilities

RESEARCH APPROACHES FOR IMPROVEMENT

• Classical / traditional approaches

• Biotechnological approaches

CLASSICAL/ TRADITIONAL APPROACHES • Germplasm collection

• Screening of germplasm

• Hybridization and selection

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

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)

Nagesh et al., 2013

Molecular characterization of cluster bean (Cyamopsis tetragonoloba L. Taub) using RAPD and ISSR 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

Identification and Characterization of EST-SSR Markers

Nagesh et al., 2014

Banding pattrens obtained from amplification of SSR markers

UDP-glucose

Pathway to galactomannan formation in plantsfructose-6-phosphate

phosphomannoisomerase (PMI)

mannose-6-phosphate

mannose-1-phosphate

mutase

PPi

GTPPPase

2Pi

GDP-mannose

GDP-mannose pyrophosphorylase

UDP-galactose

epimerase

-mannan synthase

-man-man-man-man--man-man-man-man-

gal

gal-galactosyltransferase

UDP

sucrose

UDP

fructose

hexok

inas

e

-mannan

galactomannan KSD/

020503

IDENTIFICATION OF GENES

Mannan synthase (Dhugga et al., 2004)

Galactomannan galactosyltransferase (Edwards et al., 1999)

α-galactosidase (Reid and Meier, 1973)

Genes involved in gum synthesis

Genes responsible for stress tolerance

?????

Genes responsible for plant growth and development

?????

ß-MANNAN SYNTHASE

The first plant cell wall hemicellulosic gene cloned

Dhugga et al., Science, 2004

Dr. Kanwarpal Singh Dhugga who cloned ManS gene of guar

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

Peptide mass fingerprinting of a guar protein (Unpublished data from our lab)

In our lab cloning, expression, purification and characterizationof guar plant proteins is in progress

Studying of the pathway responsible for stress tolerance in guar using the physiological, biochemical and molecular approaches is being carried out in our lab. Identification of genes responsible for stress tolerance is in progress.

(Unpublished data)

Genes responsible for stress tolerance in guar

POTENTIAL STRENGTH OF PROMOTERSAnalysis 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

Characterization of mannan synthase promoter from guar

REGULATION OF GENE EXPRESSION

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

Transformed guar plant showing expression of reporter gus gene in leaf tissue(Unpublished data)

In our lab work on RNAi in guar is in progress

Genetically Modified Guar Plantlets(Unpublished data)

Explants used:1.Cotyledon

2.Hypocotyl

3.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

PLANT CELL AND TISSUE CULTURE

Verma et al., 2013

In vitro regeneration of complete plantlets in a single culture in guar

Verma et al., 2013

OUR TEAM

Nagesh K A

Manisha Choudhary

Swati VermaPranita Bhatele

Umesh K. Tanwar Shalini Pareek

Kulvinder S. Gill Kanwarpal S. Dhugga

Pallavi Gahlot

Navneet Kaur Deepa Dewan Nishu Mittal Poonam Jaiswal

Shilpi Kumari

Prof. S.K. Tripathi

Prof. S.M. Sondhi

Prof. Pravindra Kumar

Sardar Tara Singh JiSardar Tara Singh JiMy MentorMy Mentor

THANK YOU

My website WWW.GSRANDHAWA.IN

My emailSHARNFBS@IITR.AC.IN