RobertMU ISC Dec 2013
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Transcript of RobertMU ISC Dec 2013
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Status of genetic diversity, in vitro propagationand genetic transformation in Parkia timoriana
(DC.) Merr.
Robert ThangjamDepartment of Biotechnology
School of Life Sciences
Mizoram University, Aizawl - 796004
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commonly known as tree bean - a
multipurpose treefamily Leguminosae and sub-family
Mimosoidae
most widespread species of Parkiainthe Indo-Pacific region that is
distributed from northeast India toIrian Jaya
locals in northeastern Indiaconsumed its green pods and seeds asa favourite vegetable
matured black kernels were storedfor future use
enormous socio-economic values andmedicinal properties
Parkia timoriana(DC.) Merr.
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Hopkins, 1994
Distribution of tree bean
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SH
HOOCNH2
+ HCHO
S
HOOC
R
NH
Cysteine FormaldehydeThioproline
condensation product of formaldehyde and cysteine
a well known antioxidantand anti-agingcompound
reported to be associated with the characteristic pungent smell
consumption contribute to the low incidence of stomach cancer insouthern Thailand
Thiazolidine-4-carboxylic acid (C4H7NO2S,thioproline):
Food Chemistry,1998
0 50 100 150 200 250
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Curcumin + 60% methanolic extract
Curcumin + water extract
Curcumin
Absorbanceat420nm
Gamma radiation dose (Gy)
Thiyl free radicals scavenging
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Protein content - kernel and the pods are 29% and 13-19%respectively. essential amino acid patterns is comparable to
FAO/WHO/UNU (1985) amino acid requirement for pre-schoolers
Good source of fuelwood burns slowly & completely
Industrial purposes easy to saw & good finish, bark (6-15% tannins) for tanning & dye
Oil fruit contain 19.6% oil
Agro-forestry tree nitrogen fixing; produce 10-12 cubic
meter of biomass in 10-15 years Ornamental tree planted as ornamental avenue tree
MULTIPURPOSE TREE
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AS TRADITIONAL MEDICINE
.
bark or skin of fruits dysentery, diarrhoea & piles
seeds as well as tender pods - stomach disorders &
regulateliver function
bark and leaves - lotions for skin diseases, ulcers &rheumatic affected parts
skin of fresh - applied on scabbies
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Bane for large scale production
A
B
CD
E
F
GH
A
B
C
D
E
Infestation with Cadra cautella(current science, 2003)
Infestation with Anoplophora glabripennis(DBT annual report 2006)
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Sl.
No.
Cultivar Site District Long Lat Elev
(masl)
1 Jiri Jiribam Imphal E 93.12 E 24.80 N 40
2 Kangchup Kangchup Senapati 93.81E 24.86 N 820
3 Narankonjin Narankonjin Imphal W 93.93 E 24.73 N 780
4 Kangpokpi Kangpokpi Senapati 93.97 E 25.15 N 1270
5 Moreh Moreh Chandel 94.14 E 24.18 N 700
Genetic characterization
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RAPD Profile of different cultivars
25 RAPD primers usedRAPD profile congruent with morphological data
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Genetic diversity among elite cultivars
10 standardized RAPD primers usedLow level of genetic diversity detected
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Analysis of variations within and among populations using ISSR
Kangchup Kangpokpi Narankonjin
Population % Polymorphicbands (PPB)
Allele number
(Ao)Effective allele
number (Ae)Neis gene
diversity (He)Shannon
index (I)Kangchup (KC)
Kangpokpi (KP)
Narankonjin (NA)
33.33
32.43
18.92
1.33
1.32
1.18
1.23
1.25
1.14
0.13
0.13
0.07
0.19
0.19
0.11Overall Gst(genetic differentiation) = 0.29
Overall Nm (gene flow from Gst) = 1.23
35 UBC ISSR primers used111 alleles analysed
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In vitro regeneration through multiple shoot formation
Plant material : Seeds from an 18 year old elite plustree
Explant: Cotyledonary node from 10 day old in vitrogerminated plant
Media: MS, WPM & Gamborg
Hormones: NAA & BAP (singly or in combinations)
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NAA
(mg/l)
BAP
(mg/l)
No. of shoots
0 0 1.00 0.00
0.5 0 6.50 0.30
1.0 0 1.60 0.16
2.0 0 1.50 0.16
0 0.5 3.00 0.14
0 1.0 3.40 0.22
0 2.0 5.80 0.20
0.5 0.5 2.10 0.10
0.5 1.5 3.70 0.26
0.5 2.5 6.60 0.30
NAA
(mg/l)
IBA
(mg/l)
No. of roots
0 0 0.00 0.00
1.0 0 1.60 0.16
2.0 0 2.70 0.15
3.0 0 2.70 0.15
0 0.5 2.50 0.16
0 1.0 3.20 0.13
0 2.0 8.70 0.33
Regeneration of transformants through multiple shoot production (MS)
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Bacterial strain
Agrobacterium tumefaciens strain EHA105 harboring thebinary vector pCAMBIA2301 containing a nptII (neomycinphosphotransferase) as selectable marker and the -
glucuronidase (GUS) gene (uidA) with an intron as the
reporter gene within the T-DNA region driven by CaMV35S promoter was used for the transformation.
Genetic transformation
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Kanamycin Selection
Shoot induction medium (SM): MSB + 0.5 mg/l NAA + 2.5 mg/l BAP) containingdifferent concentrations of kanamycin (0, 50, 75 and 100 mg/l)
Root induction medium (RM): MSB + 2.5 mg/l IBA supplemented with differentconcentrations of kanamycin (0, 1, 5, 7.5 and 10 mg/l)
Influence of cefotaxime on the shoot induction and subsequent growth was
also checked by culturing explants on SM and RM medium containing 500 mg/lcefotaxime.
Concentration
(mg/l)
Percentage (%)
survival explants
Shoot regeneration per
explant (%)
Average number
of shoots/explant
SE
0 100 45/45 (100) 6.07 0.28
50 77.14 27/45 (77.14) 1.4 0.29
75 11.11 5/45 (11.11) 0.33 0.12
100 0.00 0/45 (0.00) 0.00 0.00
F t ff ti t f ti ffi i
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1. Optimization of co-
culture duration (days)
GUS analysis
(Days) Percentage (%) of GUS+
plants (Mean SE)
Control 0.00
0.001 30.00 1.44
2 41.94 1.00
3 66.67 2.20
4 20.00 1.44
Factors affecting transformation efficiency
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0 min 15 min 30 min 45 min 60 min
0
10
20
30
40
50
60
%
ofGUS+Plan
ts
Period of Inoculation
2. Duration of inoculation period (Minutes) Percentage (%) of GUS+plants (Mean SE)
Control 0.00
15 21.94 1.00
30 54.72 1.21
45 43.61 0.73
60 35.64 2.18
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3. Concentration of acetosyringone
Acetosyringone
concentration
(M)
Percentage (%) of
GUS+ plants (Mean
SE)
Control 0.00 0.00
0 36.39 0.73
50 47.22 1.47
100 57.78 2.65
200 64.44 1.54
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Regeneration of transgenic plant
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Molecular analysis of transformed plants
PCR analyses showed amplification of the 540 bpfragments corresponding to the nptIIgene
540 bp
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Southern analysis
Southern blot analysis of genomic DNA of transformed and non-transformed (control) plants.
Lane 1 -Hind III digested Lamda DNA ,lane 2 - DNA from untransformed plant (control),lanes 3,4 - transformed plants,lane 5 plasmid DNA (pCAMBIA2301) of size 11.6 kb
f h f f l d d l f P
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Total no. of
explants
inoculated withAgrobacterium
Total no. of shoots
regenerated on
selection mediuma(no. of explants
showing multiple
shoots)
Total no. of shoots
rooted on
selection mediumb
Percentage of
plants established
in soil
Percentage of
plants positive for
nptII by PCR
275 825 (125) 225 60 (135/225) 33.33 (5/15)
Summary of the transformation of cotyledonary node explants of P. timorianacocultured with Agrobacterium tumefaciens strain EHA105 harbouring binaryvector pCAMBIA 2301
aSelection medium for shoot regeneration: MSB + 0.5 mg/l NAA + 2.5 mg/l BAP + kanamycin (75 mg/l) +cefotaxime (500 mg/l)bSelection medium for root induction: MSB + 2.0 mg/l IBA + kanamycin (7.5 mg/l) + cefotaxime (500mg/l)
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Summary
genetic status of tree bean in Manipur established
gene pool status at the population level worked out
techniques for in vitro regeneration established
genetic transformation protocol using Agrobacterium
established (candidate gene?)
Strategy for conservation & sustainable
production as a policy program needed
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Thank you