Zeeshan.Presentation final

BY
MD. ZEESHAN ALI
ORGANOGENESIS AND SOMATIC EMBRYOGENESIS
IN TRIGONELLA FOENUM GRAECUM
UNDER THE SUPERVISION OF
Dr ALKA NARULA HAMDARD UNIVERSITY
NEW DELHI

Fenugreek (Trigonella foenum-graecum L.) is an annual forage legume,
an important aromatic medicinal plant that is commercially used as spices
in major parts of the world. It belong to Fabaceae.
Medicinal importance: The seeds and leaves are anti-cholesterolemic,
anti-inflammatory, anti-tumor and hypoglycaemic.
The plant is known for its superior qualities of pharmaceutically and
nutraceutically rich in several phytochemicals such as carbohydrates, amino
acids, alkaloids, steroids and saponins.
Seed is of commercial interest as it is a source of diosgenin (present in
embryo in higher amounts than in endosperm), but its content is
relatively low for economical and beneficial exploitation. The diosgenin
content of seed fluctuate between1-2 %.
In addition to common breeding approaches, modern tissue culture or
in vitro techniques (use of juvenile explant) offers an important
opportunity to improve the plant secondary metabolite in Fenugreek.

Objectives of the study
1. Micropropagation of Trigonella foenum graecum via:
(a) Organogenesis
Explants: (i) Hypocotyl
(ii) Cotyledonary leaf
(b) Somatic embryogenesis
Explants: (i) Hypocotyl
(ii) Cotyledonary leaf
2. Determination of genetic fidelity through:
RAPD analysis

Organogenesis
Explants: Seed
Germination: MS Basal/ MS +GA3 (2/5 ppm)
Hypocotyl and cotyledonary leaf from seedlings were inoculated on:
Medium: 1. MS basal
2. MS + in mg/l IAA (0.5) + Kn (1) +Ad (40) + CH (500)
Somatic embryogenesis
Explant: Hypocotyl and cotyledonary leaf from seedlings
Liquid Medium: Induction- a. MS + 2, 4-D (1 mg/l)
b. MS + 2, 4-D (2 mg/l)
c. MS + 2, 4-D (5 mg/l)
SE development- 1. MS+(in mg/l) NAA(0.5)+BA(2)+GA3(2)+Put(0.5)
2. MS+(in mg/l) NAA(0.5)+BA(2)+GA3(2)+Put(2.0)
3. MS+(in mg/l) NAA(0.5)+BA(2)+GA3(2)+Put(5.0)
The morphogenic response was monitored periodically
MICROPROPAGATION

Genomic DNA was isolated by Doyle and Doyle (1990) method
RAPD analysis
Primer Nucleotide sequence (5/_3/) Annealing Temperature (ºC)
OPN-07 TCAGCCCAGAG 36
OPN-08 CACCTCAGCTC 36
OPN-09 TTGCCGGCTTG 36
List of primers used:
PCR
1.Reaction volume (15µl):
1 . 1.5µl Taq buffer (10x)
2. 1.5 µl MgCl2 (25 mM)
3. 0.8 µl dNTP’s (10 mM)
4. 1.2 µl primer (10 mM)
5. 1.2 µl Taq polymerase (1U)
6. 3.0 µl DNA (25 ng/µl)
7. 5.8 µl autoclaved double distilled water
2. Amplification process:
DNA amplification was performed in thermal cycler (eppendorf). After initial cycle of denaturation at
94°C for 4 min, 35 cycles of denaturation at 94°C for 1 min, annealing at suitable temperature (Primer
dependant) for 1 min and extension at 72°C were provided, which was followed by a final extension of
72°C for 10 min. Amplified fragments were resolved on 1.2% agarose gel using 0.5X TBE. The results of
RAPD were documented using gel documentation system (UVP- Germany).

Medium:
1. MS basal

0
10
20
30
40
50
60
70
80
90
100
4 weeks 8 weeks 12 weeks
PercentCallusing
Age
Trigonella foenum graecum: Perecnt Callusing from cotyledonary leaf
Medium 2
Medium 3
medium 4
Medium:
1. MS basal: No response
2.MS + in mg/l IAA (0.5) + Kn (1) +Ad (40) + CH (500)

0
1
2
3
4
5
6
7
8
9
10
Medium 2 Medium 3 Medium 4
Numberofshootsperexplante Trigonella foenum graecum: Number of shoots per explant
regenerated from cotyledonary leaf explant
4 weeks
8 weeks
12 weeks
Medium:

0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
4 weeks 8 weeks 12 weeks
Lengthofshoot(cm)
Age
Trigonella foenum graecum: Length of shoot (cm) per explant
regenerated from cotyledonary leaf explant
Medium 2
Medium 3
Medium 4
Medium:

0
10
20
30
40
50
60
70
80
8 weeks 12 weeks
PercentSomaticembryogenesis
Age
Trigonella foenum graecum: Percent Somatic embryogenesis
Hypocotyl
Cotyledonary leaf

0
2
4
6
8
10
12
14
16
18
8 weeks 12 weeks
Numberofsomaticembryosperexplant
Age
Trigonella foenum graecum: Number of Somatic embryos
differentiated per explant
Hypocotyl
Cotyledonary leaf

Seed Germination on MS basal medium

1 2
Explant: Hypocotyl Result: Friable Callus Age: 4 weeks
Medium: 1. MS Basal 2. MS + in mg/l IAA (0.5) + Kn (1) +Ad (40) + CH (500)

d
3
Explant: Hypocotyl Result: Friable Callus Age: 4 weeks
Medium: 3. MS + in mg/l IAA (0.5) + Kn (2) +Ad (40) + CH (500
4

Explant: Cotyledonary leaf Result: Shoot Regeneration
Medium: 1. MS basal: no response
32

Explant: Cotyledonary leaf Result: Shoot Regeneration
Medium: 4. MS + in mg/l IAA (0.5) + Kn (5) +Ad (40) + CH (500)
4a. Shoot elongation on the same medium
4 4a

b
a
c
Explant: Hypocotyl Result: Friable and Compact callus Age: 4 weeks
Liquid Induction Medium a. MS + 2, 4-D (1 mg/l)
b. MS + 2, 4-D (2 mg/l)
c. MS + 2, 4-D (5 mg/l)
Semisolid Medium: MS + in mg/l NAA (0.5) + BA (2) + GA3 (2) + Put (0.5)
BEST
Results from Hypocotyl explant

Explant: Hypocotyl Result: Compact callus Age: 4 weeks
b. MS + 2, 4-D (2 mg/l)
c. MS + 2, 4-D (5 mg/l)
Semisolid Medium: MS + in mg/l NAA (0.5) + BA (2) + GA3 (2) + Put (2)
a
b
c

C
b
a
Explant: Hypocotyl Result: Compact callus Age: 4 weeks
b. MS + 2, 4-D (2 mg/l)
c. MS + 2, 4-D (5 mg/l)
Semisolid Medium: MS + in mg/l NAA (0.5) + BA (2) + GA3 (2)) + Put (5)

Explant: Cotyledonary leaf Result: Friable and Compact callus Age: 4 weeks
b. MS + 2, 4-D (2 mg/l)
c. MS + 2, 4-D (5 mg/l)
a
b
ca
Results from Cotyledonary leaf explant
BEST

Explant: Cotyledonary leaf Result: Compact callus Age: 4 weeks
b. MS + 2, 4-D (2 mg/l)
c. MS + 2, 4-D (5 mg/l)
a
c
b

Explant: Cotyledonary leaf Result: Compact callus Age: 4 weeks
b. MS + 2, 4-D (2 mg/l)
c. MS + 2, 4-D (5 mg/l)
C
b
a

Explant: Cotyledonary leaf Result: Somatic embryogenesis Age: 8 weeks

M 1 2 3 4 5 6 7 8 9
2.0 Kb
0.94 Kb
0.56 Kb
RAPD amplifications of Trigonella foenum graecum with primer OPN-07
M = Marker digested with Hind III and Eco R1, 1= Mother plant, 2-9 = Regenerants

M 1 2 3 4 5 6 7 8 9
2.0 Kb
0.94 Kb
0.56 Kb

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Micropropagation of Trigonella foenum graecum
Clonal fidelity of regenerants
In vitro raised plantlets are genetically uniform as revealed by
RAPD analysis
1. Organogenesis
(a) Hypocotyl
(Result: Callusing)
(b) Cotyledonary leaf
Best Medium (Medium 2)
MS + in mg/l IAA (0.5) + Kn (1) +Ad (40) + CH (500)
Best Medium (Medium 4)
MS + in mg/l IAA (0.5) + Kn (5) +Ad (40) + CH (500)(Result: Regeneration)
2. Somatic embryogenesis
(a) Hypocotyl/ (b) Cotyledonary leaf
Best Medium for induction (Medium a)
MS + 2, 4-D (1 mg/l)
Best Medium for SE development (Medium 1)
MS + in mg/l NAA (0.5) + BA (2) +GA3 (2) + Put (0.5)

I take the opportunity to express my deep sense of indebtedness and sincere gratitude
to Dr Alka Narula for her constant encouragement and interaction that made my work
more fun and enjoyable.
I extend my thanks to all the teachers in the Department of Biotechnology.
It gives me an immense pleasure to express my thanks to the research scholars
Ms Kshipra and Mr Sumit for the help provided to me.
I am thankful to the technical staff especially Mr. Anish Cherian, Mrs. Noor Fatima
and Mrs. Sarika. I acknowledge the help provided by lab helping staff that includes
Mr. Gautam, Mr. Taufiq and Mr. Sajid.
Last but not the least I would like to thank my family & friends for support.
Especial thanks goes to my friend Md Zia Ulla Hasmi who was always with
me while doing the dissertation related work.
ACKNOWLEDGEMENT

Zeeshan.Presentation final

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