Establishment of efficient transformation system in cicer arietinum with gus gene

PRIYA GOSWAMI
4th Semester
Plant Physiology Biochemistry & Plant Molecular
biology Special Paper , 2013

Why Chickpea?
 Cicer arietinum (Common Name :- Chickpea) is an
important food crop in much of the developing countries.
 Ranks third in production among food legumes.
 It is good as a source of carbohydrate (48.2 - 67.6%),
protein (12.4 - 31.5%), fat (6%) and nutritionally important
minerals.
 This pulse crop is an important source of dietary protein
for human consumption, it is also important for the
management of soil fertility due to its nitrogen-fixing ability
 Provides excellent quality of protein at affordable prices to
the poor and average income families.
Fig 1 : Chickpea
plant
*SYSTEMATIC POSITION*
Kingdom : Plantae
Order : Fabales
Family : Fabaceae
Sub-family: Faboideae
Genus: Cicer
Species: C. arietinum
•Binomial name: Cicer arietinum L.

Short Description Of Chickpea
Plants
Fig 2 : Different Parts Of Chickpea
Plant
Leaves
Flower
Pod
 Chickpea is an annual plant with plant height
ranging between 30-70cm.
 The plant has a deep root system with
nodules & thus very efficient in fixing
atmospheric nitrogen.
 They have compound leaves which are
arranged in alternate phyllotaxy and are
generally imparipinnate with 11-13 leaflets
and covered with glandular hairs.
 Flowers are solitary axillary and are generally
arranged in inflorescence of two to three. They
are white, pink, purplish, or blue in colour.
 Chickpeas are a type of pulse with one
seedpod containing two to three peas.

Fig 3 : Different Coloured Flowers Of Chickpea
Fig 4: Chickpea Pod
Fig 5: Chickpea Pod Containg Peas

Origin & Cultivation
 ORIGIN & CULTIVATION : Chickpea is probably
originated in an area of south-eastern Turkey and
adjoining Syria, but is now cultivated throughout the
semi-arid regions of the world.
Fig 6: Cultivation Of Chickpea Throughout The World
Primary Zone Of Chickpea Production
Secondary Zone Of Chickpea Production
Tertiary Zone Of Chickpea Production
 CHICKPEA PRODUCTION IN INDIA : India is the
largest producer and consumer of chickpea in the
world, and accounts for more than 60% of the area
harvested and of the total worldwide production.
Fig 7 : Different Zones Of Chickpea Production In India

 GROWING CONDITIONS :
 TYPES :
TEMPERATURE 18-26°C @ day and
21-29°C @ night
RAIN FALL 600-1000 mm
Fig 8: Desi Fig 9 : Kabuli
Small, angular
seeds that are
variously
pigmented and
are grown
principally on the
Indian
subcontinent and
in East Africa
Relatively large,
round seeds of
white or pale cream
colour and are
predominantly
grown in the
Mediterranean
region and in Central
and South America.
Growing Conditions & Types

Nutritional Content & Uses
Fig 10 : Pie Chart For The Nutritional Content Of Chickpea
Carbohydrate (with
47% starch)
Protein (with 8%
crude fibre)
Fat
P,Fe,ash &other
water soluble
vitamins
 NUTRITIONAL CONTENT :
 USES :
AS FOOD AS MEDICINE OTHER USES
Due to high protein, high
unsaturated fatty acid,
cellulose, calcium, zinc,
potassium, high amount of
vitamin- B content chickpeas
are used as food.
• Chickpeas contain isoflavone
which has a big influence on the
health of women, their estrogenic
activity & also prevents the
proliferation of cancer cells
causing cancer cell death.
• They also contain chromium
which plays an important role in
glucose & lipid metabolism.
• Animal feed.
• Preparation of adhesive.
• Preparation of dye.
• Preparation of vinegar.
• Cooked chickpea-milk mixture
is good for feeding infants,
effectively controlling diarrhoea.
• Used for textile sizing.

AFFECTED
CHICKPEA
YIELD
BIOTIC
STRESS
ABIOTIC
STRESS
THESE ARE MAINLY
DISEASES eg:
 DRY ROOT ROT
 BOTRYTIS GREY MOLD
 COLLAR ROT
 FUSARIUM WILT
eg:
DROUGHT
SALINITY
LOW TEMPERATURE
Main Problems In Chickpea
Production

AFFECTED
CHICKPEA
YIELD
BIOTIC
STRESS
ABIOTIC
STRESS
THESE ARE MAINLY
DISEASES eg:
 DRY ROOT ROT
 BOTRYTIS GREY MOLD
 COLLAR ROT
 FUSARIUM WILT
eg:
DROUGHT
SALINITY
LOW TEMPERATURE
Main Problems In Chickpea
Production
Diseases are the most
serious constraints of
chickpea production

Some Common Diseases In Chickpea

Aims & Objective Of My Project
Tissue culture and transformation in chickpea using gus gene governed by i.
Constitutive Actin1 Promoter, ii Pod specific Promoter.
•In vitro germination of surface sterilised chickpea seeds for embryo axis
development using MSO media.
•Agrobacterium mediated transformation of the developed explants.
•Selection of the plants in hygromycin.
•Hardening of the selected transformants in the soil.
•PCR analysis of the transgenics .
.

•In vitro germination of surface sterilised chickpea seeds for
embryo axis development using MSO media.
.

Fig 11 : Chickpea Seeds Of
Variety DCP-92-3
Fig 12 : Imbibition Of
Chickpea Seeds
Fig 13: Surface Sterilized
Seeds Were Plated In
MSO* Plates After
Removal Of The Seed Coat
Fig 14 : 2 Day Grown
Seeds In MSO* Media
Fig 15 : 3 Day Grown
Seeds In MSO* Media
Some Seeds Were Further
Used For Transformation
Fig 16 : Control Plants Were Grown In
The Greenhouse Condition*
in vitro germination of surface sterilised chickpea seeds for embryo axis
MSO*= Murashige & Skoog
Basal Media (1962)
Greenhouse Condition*= with
16/8 (light/dark) photoperiod
temp. 20°C under high
humidity with frequent
watering.

•in vitro germination of surface sterilised chickpea seeds for embryo axis
•Agrobacterium mediated transformation of the developed
explants.
.

Fig 15 : 3 Day Grown Seeds In MSO Media
Are Used As Explants For Transformation
Agrobacterium
LBA4404 with
vector
containing Pod
Specific
Promoter
Agrobacterium
LBA4404 with
vector containing
Actin1 Constitutive
Promoter
Fig 17: 20 ml Agrobacterium Culture In LB Media With Rifampicin & Kanamycin
Antibiotic & kept In the Incubator Shaker @ 28°C & 120 rpm For 2 Days
O.D. Value Of 2day Old Agrobacterium Culture Was Measured (i.e.
approximately around 3.0)
The Cuture Was Pellet Down @ 3500 rpm for 20 mins At The Bottom Of
The Oakridge Tube
Pellet was dispersed in 40ml Infiltration Media (MS20*)+ 40µl
Acetosyringone & poured in the petriplates
Explants Were Pricked With Injection Syringe
Then Transferred To The Petriplate For
Agrobacterial Infection.
The Petriplate Was Kept In The Vacuum Desiccator
For 20 minutes. The Pressure Is Maintained at 600
mm of Hg
Agrobacterium mediated transformation of the developed
explants.
After 20 mins The Explants Were Blotted
Dry & Transferred To Petriplates
Containing MSO-As* Media & Covered With
Aluminium Foil & Kept In The Incubator @
28°C For 2 days.
MS20*=MS basal media with 20g/l sucrose
MSO-As*=MS basal media with
acetosyringone
Fig 18: Vacuum Desiccator

Vector Construct Used For
Transformation
Fig 20: pCAMBIA 1301 (-) GUS Vector -Fig a: Containing Actin1
Constitutive Promoter; Fig b: Containing Pod Specific Promoter
LB = Left Border
RB = Right Border
Actin1 = Constitutive Promoter
pds= Pod Specific Promoter
nos = Nopaline synthase
hpt = Hygromycin phosphotransferase.
gus = Reporter Gene
nptII= neomycinphosphotransferase II
Fig 19 : Vector map of pCAMBIA 1301
Fig: a
Fig: b
nptII
nptII

•in vitro germination of surface sterilised chickpea seeds for embryo axis
.

Selection Of The Plants In Hygromycin.
After 2 days from the
Agrobacterial infection,the explants
were transferred to Magenta boxes
containing selection media with
hygromycin (50 mg/l) for plant
selection.
The transformed explants can be
easily distinguished from the non
transformed ones on the basis of
their viability in the medium.
Fig 21: Transformed Plants In Hygromycin
Selection Media
Fig a:-With Constitutive Actin1 Promoter
Fig b:-With Pod Specific Promoter
b.
a.

Hardening of the selected transformants in the
soil
 Rooted plantlets with varying shoot
lengths were carefully taken out of the
magenta boxes and roots were washed
thoroughly in water to remove the agar
and placed in the soil and then they
were kept in the greenhouse with
containment facility, maintained with
16/8 (light/dark) photoperiod,20°C
temperature,under high humidity with
frequent watering.
Fig 22 : Transformed plants with GUS gene a. Governed by
Constitutive promoter b. Governed by Pod Specific
Promoter -were grown successfully in soil.
a. b.

PCR analysis of the transgenics.
Fig 23 :Gel Showing PCR Products Of The Genomic DNA Of Chickpea
L1,L2,L3: Transformed Plants with Pod Specific Promoter
L4,L5: Transformed Plants with Actin1 Promoter
L6:Negative Control (Non Transformed Plant)
L7:Positive Control Plasmid
Result: Distinct bands were observed in L2,L3,L4,L5,L6.Thus
the PCR analysis showed the presence of gus gene.
567 bp
(Amplified gus)
(Approx.)
L1 L2 L3 L4 L5 L6 L7

TABLE 1: SHOWING THE TRANSFORMATION FREQUENCY OF
CHICKPEA
CULTIVAR OF
CHICKPEA
PROMOTER USED FOR
TRANSFORMATION
NO. OF SEEDS
PLATED
NO. OF SEEDS
GERMINATED
NO. OF PLANTS
TRANSFORMED
% OF TRANSFORMED
PLANTS WITH
RESPECT TO TOTAL
NO. OF SEEDS
% OF TRANSFORMED
PLANTS WITH
RESPECT TO
GERMINATED PLANTS
DCP-92-3 Actin1 47 41 4 8.51% 9.75%
DCP-92-3 Pod Specific 156 139 25 16.02% 17.99%
0
50
100
150
% Of Germination % Of Transformation Of@ Total
Seeds
% Of Transformation Of@
Germination
Graph Showing Germination & transformation Efficiency
DCP-92-3 (With Actin1 Promoter) DCP-92-32 (With Pod Specific Promoter)

Histochemical Staining
• From the regenerated explants, the
section of the seeds were excised
using a sterile scalpel and were
immersed in GUS staining solution.
• Then the excised explants in the
staining solution were incubated at
37°C for 24 hours.
Fig 24 : gus expression
a: For Transformed Plants (with Actin1 promoter)
B: For Wild Type Plants
a. b.

Thus in this study ,the following have been accomplished-
i. Development of efficient regeneration system has been studied and
experimentally standardized.
ii. Agrobacterium mediated gene transformation protocol for chickpea
variety DCP-92-3 has been developed with gus gene &
iii. Transient gus expression has been confirmed by the presence of
specific bands in PCR & through histochemical staining.
 So transformation of pCAMBIA 1301 containing the gus gene into
the chickpea variety DCP-92-3 was successfully done in my project.
Conclusion

Acknowledgement
At the very beginning, I feel highly privileged to extend my sincere gratitude to my respected supervisor Dr.
Karabi Datta for suggesting me this interesting project work. It is my privilege that I got the opportunity to work
under her benevolent guidance.
Moreover, I would like to thank Prof A.K Biswas who has always helped and motivated me during my project
work.
I would also like to convey my deepest gratitude and sincere regards to Dr Sailendra Nath Sarkar for his
continuous support, useful advice and encouragement in completing my project work.
I would also wish to acknowledge with sincere appreciation and deepest gratitude to my co guide Dr. Moumita
Ganguly who has guided me at every step and has helped me immensely throughout this project. I am also
thankful to Ms. Sayani Majumder for her constant help and support.
The content of the text, pictures, figures and protocols are incorporated in this review based on the literature
available in the text books, E-survey and various Research papers.
Last but not the least; I am ever thankful to all the scholars of Plant Molecular Biology and Biotechnology
Laboratory for their constant suggestions and kind guidance during the course of my project work.
Overall it was a great learning experience and I am indebted to my friends and family for their support and to
everyone with whomever I have had the opportunity to work with.

Establishment of efficient transformation system in cicer arietinum with gus gene

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