RobertMU ISC Dec 2013

8/13/2019 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

RobertMU ISC Dec 2013

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