International Jatropha Conference, IPB International Convention Center Bogor

Indonesia, June 24th-26th, 2008

The Development of Agrobacterium--mediated

Transformation Procedure of Jatropha curcas L.

Sri Nanan Widiyanto

srinanan@sith.itb.ac.id

Plant Sciences and Biotechnology

School of Life Sciences and Technology

Institut Teknologi Bandung

 Background

Jatropha curcas L.:

A dedicated energy crop
Produces biodiesel oil-bearing
seeds

Problems:
Required genetic improvement for the future needs

S.N.Widiyanto-ITB
 Agrobacterium-mediated transformation

Passing the long period required for natural

genetic crosses and selection
Introduction of genes encoding desirable

Application of genetic transformation procedure

(Li et al., 2007)
Using Agrobacterium tumefaciens as a mediator
Introduce phosphinothricin acetyltransferase (bar) and ß-
glucuronidase (uidA) genes
Using cotyledon discs
S.N.Widiyanto-ITB
Consideration in application

To overcome the technical difficulty of

transclone regeneration:

Using regenerative tissues of plant organs,

pre-existing shoots and single-node cuttings
(Datta et al., 2007)
 The objective of research

to develop the procedures of Agrobacterium--

mediated transformation and in vitro regeneration of
Jatropha curcas L. using embryonic tissues of
mature embryos and non-embryonic tissues of
nodal and internodal segments which were excised
from in vitro regenerated shoots
Research activities

Sensitivity explants to antibiotics

In vitro regeneration
Transformation
Evaluation of transformation efficiency
In vitro regeneration of transclones
MATERIALS AND METHODS
 Plant Materials

Embryos were isolated from surface sterilized seeds of J. curcas:

Initial explants for developing procedures of in vitro regeneration
Target tissues in Agrobacterium--mediated transformation

Cotyledonary nodal cuttings were excised from seedlings:

Initiate axillary shoot proliferation
Source for nodal and internodal cuttings

Nodal and internodal cuttings:

Explant sources in in vitro propagation
Target tissues in Agrobacterium--mediated transformation

In vitro cultures condition:

In a culture room at 25 ± 2ºC
Continuous light condition with 40 μmol m-2s-1
Plant Materials
 Bacterial Cultures

(Jefferson et al., 1987)

Agrobacterium tumefaciens strain LBA-4404

pBI-121 binary vector carrying uidA (-glucuronidase/GUS)
nptII (neomycin phosphotransferase II for kanamycin resistance)
A. tumefaciens LBA-4404 without binary vector as the control
treatment

Bacteria preparation:
YEB medium supplemented with 100 mg/l streptomycin, 100 mg/l
rifampicin and 50 mg/l kanamycin for selection of the pBI-121 vector
Agrobacterium filtrates cultured at 28oC over night before being used
Expression of uidA gene used as a visual marker
Expression of nptII gene used as a selectable marker
Culture Media

In vitro propagation:
B1 Medium:
MS medium (Murashige & Skoog, 1962)
0.5 µM N6-benzylaminopurine (BA)
0.5 µM α-naphthalene acetic acid (NAA)

B2 Medium
MS medium (Murashige & Skoog, 1962)
0.5 µM N6-benzylaminopurine (BA)
1.0 µM α-naphthalene acetic acid (NAA)
Culture Media

Antibiotic sensitivity tests:

Growth regulator free MS medium
25 - 100 mg/l kanamycin
50 - 300 mg/l amoxicillin

Selective medium:
B1 Medium or B2 Medium
50 mg/l kanamycin
200 mg/l amoxicillin

All culture media:

pH 5.8 (drops of 1 N NaOH or 1 N HCl)
0.25% Gelrite to solidify
Autoclaved at 121oC, 1.2 kg cm-2 for 15 min
 Procedure

Sensitivity Explants to Antibiotics

Embryos
Growth regulator free MS medium with kanamycin at
25 - 100 mg/l or
Growth regulator free MS medium with amoxicillin at
50 - 300 mg/l
Growth of seedlings observed 3 week-subculture
period
Healthy performance: normal, greenish seedlings
Lethal toxicity response: evidence of light to dark
brown necrotic tissues in seedlings
 Procedure

In Vitro Regeneration:
Optimizing medium for axillary shoot growth and proliferation
Cotyledonary shoots without roots from 8-10 day-old
seedlings
Growth regulator free MS media to initiate shoot elongation
Cotyledonary nodal-cuttings (8-10 mm) cultured on B1 Medium
or B2 Medium
Growth parameters:
Numbers of elongated axillary shoots
Shoot length (mm)
numbers of nodes of elongated axillary shoots per node
Selected regeneration medium used to regenerate J. curcas
transclones
 Procedure

Inoculation and Co-cultivation (Charity et al., 2002):

Sterile embryos, nodal & internodal segments pre-conditioned
in liquid growth regulator free MS medium for 24 hr
Infiltration:
Pre-conditioned explants
vacuum infiltrated at -80 kPa for 5 min.
Suspension culture of A. tumefaciens (OD600nm = 0.6-0.8)
Vacuum-infiltrating was conducted in a sterile dessicator
Inoculated J. curcas explants
Co-cultivated:
on solid, growth regulator free MS medium
in dark condition at 25 ± 2ºC, three days
 Procedure

After co-cultivation:
Co-cultivated explants washed with sterile distilled water
Immersed in 300 mg/l amoxicillin solution for 10-20 min
Plotted to dry on a sterilized filter paper
Collected for the following experiments

Inoculated explants for:

Histochemical GUS analyzing on day 3 after co-cultivation
Cultured on solid, selective MS medium + 50 mg/l
kanamycin + 200 mg/l amoxicillin
Procedure

Evaluation of transformation efficiency

Transformation efficiency estimation:
Percentage of inoculated explants
Showed GUS expression confirmed by using the
GUS histochemical assay (Jefferson et al., 1987)
Selected transclones on medium MS with kanamycin

Percentage values:
Numbers of selected explants performing
expressions of marker genes per total numbers of
evaluated explants of each treatment x 100%
RESULTS
 Sensitivity of explants to antibiotics

After three week-subculture period:

Seedlings performed greenish, healthy performance
and normal growth on culture medium containing
less than 50 mg/l kanamycin or 200 mg/l amoxicillin
(data not shown)
On medium containing more than 50 mg/l
kanamycin or 200 mg/l amoxicillin, the evidence of
light to dark brown necrotic tissues was indicated
on tissues of seedlings
In the following experiments selective medium was
added with kanamycin at 50 mg/l and amoxicillin at
200 mg/l
In Vitro Regeneration
Table 1.
Effects of B1 Medium and B2 Medium on the growth of axillary
shoot-buds of cotyledonary nodal segments of J. curcas.

Growth Parameter Regeneration Media*)

Evaluated
B1 Medium B2 Medium
Average of no. of shoots 3.8 ± 0.8 3.9 ± 0.6
per nodal segment
Average of shoot length 16.6 ± 1.2 14.8 ± 1.8
(mm)

B1 Medium was MS medium with 0.5 µM BA and 0.5 µM NAA

B2 Medium was MS medium with 0.5 µM BA and 1.0 µM NAA
Shoot Multiplication
 Percentages of GUS positive
100 90.9
90
80 75.2
63.6
explants (%)
70
60 53.9 55.2
46.9
50
40
30
20
10
0
Embryos Nodes Internodes
Explant sources

Day 3 Day 14

Fig. 1. Percentages (%) of inoculated embryos, nodal and

internodal segments with GUS positive expression on day
3 and day 14
 100 90.9
Percentages of GUS positive
87.01
90
expression (%) 80
70 65.54
60
50
40
30
20
10
0
Embryos Cotyledons Radicles

Explant parts

Fig. 2. Percentages (%) GUS positive expression of embryos

and its parts on day 3
 100
Percentages of GUS positive 89
90
80 72.9
expression (%)
70 62.5
60
50
40
30
20 10.1
10
0
Selected seedlings Cotyledons Hypocotyls Roots

Seedling parts

Fig. 3. Percentages (%) GUS positive expression of selected

seedlings and its parts on day 3
 Concluding Remarks

Embryonic and non-embryonic tissues of J. curcas

were potential as the target explants in
Agrobacterium-mediated transformation
Efficiency of transformation was varied between
embryonic and non-embryonic tissues
Compatibility of A. tumefaciens strain LBA4404 with
J. curcas explants was tissue-specific
Selected transclones from nodal cuttings were able
to regenerate on selective medium
 Acknowledgments

I thank Heni Rahmania, Fajar D. Arumsari, Perswina

Allaili, Annisa Ramdhaningtias, and Prita Purwijanarti
at School of Life Sciences and Technology, ITB,
Bandung

This study was partly supported by the ITB Research

Fund 2006 – 2007
SK 0018/K01.03.2/PL2.1.5/I/2006
SK 0017/K01.03.2/PL2.1.5/I/2007
Laboratory Facility - ITB

S.N.Widiyanto-ITB