Transcript Dr. G.S. Randhawa, Department of

Increasing productivity of
guar and guar gum
G. S. Randhawa
Department 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)
Molecular characterization of cluster bean (Cyamopsis tetragonoloba
L. Taub) using RAPD and ISSR markers
Nagesh et al., 2013
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
Pathway to galactomannan formation in plants
sucrose
UDP
fructose-6-phosphate
phosphomannoisom
erase (PMI)
fructose
mannose-6-phosphate
mutase
mannose-1-phosphate
UDP-glucose
GDP-mannose
pyrophosphorylase
epimerase
PPase
2Pi
b-mannan synthase
UDP
gal
-man-man-man-mangal
PPi
GDP-mannose
UDP-galactose
galactomannan
GTP
-man-man-man-man- b-mannan
a-galactosyltransferase
KSD/02050
3
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
?????
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
Pioneer Hi-Bred International Inc. where ManS gene of guar was cloned
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)
Genes responsible for stress tolerance in guar
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)
REGULATION OF GENE EXPRESSION
Characterization of mannan synthase promoter from guar
POTENTIAL STRENGTH
OF PROMOTERS
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
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
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)
Genetically Modified Guar Plantlets
(Unpublished data)
PLANT CELL AND TISSUE CULTURE
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
Verma et al., 2013
In vitro regeneration of complete plantlets in a single culture in guar
Verma et al., 2013
OUR TEAM
Kulvinder S. Gill Kanwarpal S. Dhugga Pranita Bhatele
Shilpi Kumari
Umesh K. Tanwar
Manisha Choudhary
Nagesh K A
Shalini Pareek
Swati Verma
Pallavi Gahlot
Prof. S.K. Tripathi
Prof. S.M. Sondhi
Prof. Pravindra Kumar
Navneet Kaur
Deepa Dewan
Nishu Mittal
Poonam Jaiswal
Sardar Tara Singh Ji
My Mentor
THANK YOU
My website
WWW.GSRANDHAWA.IN
My email
[email protected]