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Status of genetic diversity, in vitro propagation and genetic transformation in Parkia timoriana (DC.) Merr.

Department of Biotechnology School of Life Sciences Mizoram University, Aizawl - 796004

Robert Thangjam

Parkia timoriana (DC.) Merr.


commonly known as tree bean - a multipurpose tree family Leguminosae and sub-family Mimosoidae most widespread species of Parkia in the Indo-Pacific region that is distributed from northeast India to Irian Jaya locals in northeastern India consumed its green pods and seeds as a favourite vegetable matured black kernels were stored for future use enormous socio-economic values and medicinal properties

Distribution of tree bean

Hopkins, 1994

Thiazolidine-4-carboxylic acid (C4H7NO2S,thioproline):


condensation product of formaldehyde and cysteine a well known antioxidant and anti-aging compound reported to be associated with the characteristic pungent smell consumption contribute to the low incidence of stomach cancer in southern Thailand
SH S R NH HOOC

+
HOOC NH2

HCHO

Cysteine Thioproline
1.1 1.0 0.9

Formaldehyde
Curc umin + 60% methanolic ext ract Curcumin + water ext ract Curcumin

Absorbance at 420 nm

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 50 100 150 200 250

Thiyl free radicals scavenging


Gamma radiation dose (Gy)

Food Chemistry, 1998

MULTIPURPOSE TREE
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 preschoolers Good source of fuel wood burns slowly & completely Industrial purposes easy to saw & good finish, bark (615% 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

AS TRADITIONAL MEDICINE

.
bark or skin of fruits dysentery, diarrhoea & piles

seeds as well as tender pods - stomach disorders & regulate liver function
bark and leaves - lotions for skin diseases, ulcers & rheumatic affected parts skin of fresh - applied on scabbies

Bane for large scale production

B
D

A C
D F G E
A C E

Infestation with Anoplophora glabripennis (DBT annual report 2006)

Infestation with Cadra cautella (current science, 2003)

Genetic characterization

Sl. No. 1 2 3 4 5

Cultivar Jiri Kangchup Narankonjin Kangpokpi Moreh

Site Jiribam Kangchup Narankonjin Kangpokpi Moreh

District Imphal E Senapati Imphal W Senapati Chandel

Long 93.12 E 93.81E 93.93 E 93.97 E 94.14 E

Lat 24.80 N 24.86 N 24.73 N 25.15 N 24.18 N

Elev (masl) 40 820 780 1270 700

RAPD Profile of different cultivars

25 RAPD primers used RAPD profile congruent with morphological data

Genetic diversity among elite cultivars

10 standardized RAPD primers used Low level of genetic diversity detected

Analysis of variations within and among populations using ISSR

Kangchup

Kangpokpi

Narankonjin

35 UBC ISSR primers used 111 alleles analysed

Population Kangchup (KC)

% Polymorphic bands (PPB) 33.33

Allele number Effective allele (Ao) number (Ae) 1.33 1.23

Neis gene diversity (He) 0.13

Shannon index (I) 0.19

Kangpokpi (KP)

32.43

1.32

1.25
1.14

0.13
0.07

0.19
0.11

Narankonjin (NA) 18.92 1.18 Overall Gst (genetic differentiation) = 0.29 Overall Nm (gene flow from Gst) = 1.23

In vitro regeneration through multiple shoot formation

Plant material : Seeds from an 18 year old elite plus tree Explant: Cotyledonary node from 10 day old in vitro germinated plant Media: MS, WPM & Gamborg Hormones: NAA & BAP (singly or in combinations)

Regeneration of transformants through multiple shoot production (MS)


NAA (mg/l) 0 0.5 1.0 2.0 0 0 0 0.5 0.5 0.5 BAP (mg/l) 0 0 0 0 0.5 1.0 2.0 0.5 1.5 2.5 No. of shoots 1.00 0.00 6.50 0.30 1.60 0.16 1.50 0.16 3.00 0.14 3.40 0.22 5.80 0.20 2.10 0.10 3.70 0.26 6.60 0.30

NAA (mg/l) 0 1.0 2.0 3.0 0 0 0

IBA (mg/l) 0 0 0 0 0.5 1.0 2.0

No. of roots
0.00 0.00 1.60 0.16 2.70 0.15 2.70 0.15 2.50 0.16 3.20 0.13 8.70 0.33

Genetic transformation
Bacterial strain

Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA2301 containing a nptII (neomycin phosphotransferase) as selectable marker and the glucuronidase (GUS) gene (uidA) with an intron as the
reporter gene within the T-DNA region driven by CaMV 35S promoter was used for the transformation.

Kanamycin Selection
Shoot induction medium (SM): MSB + 0.5 mg/l NAA + 2.5 mg/l BAP) containing different concentrations of kanamycin (0, 50, 75 and 100 mg/l) Root induction medium (RM): MSB + 2.5 mg/l IBA supplemented with different concentrations 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/l cefotaxime. Concentration (mg/l) 0 50 75 Percentage (%) Shoot regeneration per Average number survival explants explant (%) of shoots/explant SE 6.07 0.28 100 45/45 (100) 77.14 11.11 27/45 (77.14) 5/45 (11.11) 1.4 0.29 0.33 0.12

100

0.00

0/45 (0.00)

0.00 0.00

Factors affecting transformation efficiency


1. Optimization of coculture duration (days)
(Days) Control 1 2 3 4 Percentage (%) of GUS+ plants (Mean SE) 0.00 0.00 30.00 1.44 41.94 1.00 66.67 2.20 20.00 1.44

GUS analysis

2. Duration of inoculation period

(Minutes) Control 15 30 45 60

60

Percentage (%) of GUS+ plants (Mean SE) 0.00 21.94 1.00 54.72 1.21 43.61 0.73 35.64 2.18

% of GUS+ Plants

50

40

30

20

10

0 0 min 15 min 30 min 45 min 60 min

Period of Inoculation

3. Concentration of acetosyringone
Acetosyringone concentration (M) Control 0 50 100 200 Percentage (%) of GUS+ plants (Mean SE) 0.00 0.00 36.39 0.73 47.22 1.47 57.78 2.65 64.44 1.54

Regeneration of transgenic plant

Molecular analysis of transformed plants


PCR analyses showed amplification of the 540 bp fragments corresponding to the nptII gene

540 bp

Southern analysis

Southern blot analysis of genomic DNA of transformed and nontransformed (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

Summary of the transformation of cotyledonary node explants of P. timoriana cocultured with Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA 2301
Total no. of explants inoculated with Agrobacterium Total no. of shoots Total no. of shoots Percentage of Percentage of regenerated on rooted on plants established plants positive for selection mediuma selection mediumb in soil nptII by PCR (no. of explants showing multiple shoots)
825 (125)
a

275

225

60 (135/225)

33.33 (5/15)

Selection medium for shoot regeneration: MSB + 0.5 mg/l NAA + 2.5 mg/l BAP + kanamycin (75 mg/l) + cefotaxime (500 mg/l) b Selection medium for root induction: MSB + 2.0 mg/l IBA + kanamycin (7.5 mg/l) + cefotaxime (500 mg/l)

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

Thank you

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