the development of agrobacterium mediated transformation procedure of jatropha curcas
TRANSCRIPT
The Development of Agrobacterium-mediated
Transformation Procedure of Jatropha curcas L.
Sri Nanan Widiyanto
Plant Sciences and BiotechnologySchool of Life Sciences and Technology
Institut Teknologi Bandung
International Jatropha Conference, IPB International Convention Center Bogor
Indonesia, June 24th-26th, 2008
Background
Jatropha curcas L.:
A dedicated energy crop
Produces biodiesel oil-bearing seeds
S.N.Widiyanto-ITB
Problems:
Required genetic improvement for the future needs
Agrobacteriummediated transformation
Passing the long period required for natural
genetic crosses and selection
Introduction of genes encoding desirable
S.N.Widiyanto-ITB
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
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 medium25 - 100 mg/l kanamycin50 - 300 mg/l amoxicillin
Selective medium: B1 Medium or B2 Medium50 mg/l kanamycin200 mg/l amoxicillin
All culture media:pH 5.8 (drops of 1 N NaOH or 1 N HCl)0.25% Gelrite to solidifyAutoclaved 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. curcastransclones
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 explantsShowed 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%
Procedure
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
Evaluated
Regeneration Media*)
B1 Medium B2 Medium
Average of no. of shoots
per nodal segment
3.8 ± 0.8 3.9 ± 0.6
Average of shoot length
(mm)
16.6 ± 1.2 14.8 ± 1.8
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
Fig. 1. Percentages (%) of inoculated embryos, nodal and
internodal segments with GUS positive expression on day
3 and day 14
90.9
63.6
75.2
53.946.9
55.2
0
10
20
30
40
50
60
70
80
90
100
Embryos Nodes Internodes
Explant sources
Perc
en
tag
es o
f G
US
po
sit
ive
exp
lan
ts (
%)
Day 3 Day 14
Fig. 2. Percentages (%) GUS positive expression of embryos
and its parts on day 3
90.987.01
65.54
0
10
20
30
40
50
60
70
80
90
100
Embryos Cotyledons Radicles
Explant parts
Perc
en
tag
es o
f G
US
po
sit
ive
exp
ressio
n (
%)
Fig. 3. Percentages (%) GUS positive expression of selected
seedlings and its parts on day 3
89
62.5
10.1
72.9
0
10
20
30
40
50
60
70
80
90
100
Selected seedlings Cotyledons Hypocotyls Roots
Seedling parts
Perc
en
tag
es o
f G
US
po
sit
ive
exp
ressio
n (
%)
Concluding Remarks
Embryonic and non-embryonic tissues of J. curcaswere 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