Tissue Culture of Jatropha curcas

Tissue Culture of
Jatropha curcas
10-month Project Report (Aug. 2007 – Jan.
2008, May 1, 2008 to August 31, 2008 )
Portia Gamboa – Lapitan
Professor 7
Department of Forest Biological Sciences
College of Forestry and Natural Resources
A. Project Objectives
To develop tissue culture protocol for
the mass propagation of high oilyielding varieties of Jatropha
adaptable to the Philippines.
• sterilization scheme for tissues
for culture
• best/most responsive
tissue/explant for culture
• appropriate medium for callus
formation, shoot induction and
rooting
• incubation or culture conditions
(light, temperature, photoperiod)
for growth and development of
Jatropha in culture
• best time for plantlets to be
outplanted from the culture vessels
B. Methodology
Selection of sources of tissues for
culture
Jatropha PGL ‘07
Selected and graded seeds of Jatropha used as
source of explants
Jatropha PGL ‘07
Sources of explants were also selected from
plants in the nursery and field
Jatropha PGL ‘07
Mature plants of Jatropha for explants
collection were also selected
Selected plants are sectioned and cultured in
different culture media for organogenesis/plantlet
development
Jatropha PGL ‘07
Activated charcoal in the culture medium
facilitates germination of Jatropha seeds
Jatropha Tissue Culture
C. Accomplishments and Major
Findings
1. Sterilization scheme for Jatropha
Table 1. Efficacy of sterilization schemes used for Jatropha
tissue cultures
Total No. of
Ave. No. of Cultures
Ave. Percent
Sterilization Scheme
Cultures
Contaminated
Fungal
Bacterial
Contamination
Fungal
Bacterial
5% calcium hypochlorite for 10 minutes
•
Seeds
20/trial of 10
trials
2/trial
1/trial
10%
5%
Leaf Tissues
10/trial of 10
trials
5-6/trial
1/trial
50-60%
10%
Nodal sections
5/trial of 10 trials
3/trial
10/trial of 10
trials
6-7/trial
1/trial
60-70%
10%
Shoot tip
5/trial of 10 trials
2/trial
1/trial
40%
20%
Young nodal sections
5/trial of 10 trials
3/trial
2% Manzate for 30 min
5% calcium hypochlorite for 20 minutes
60%
2% Manzate for 20 min
5% calcium hypochlorite for 10 minutes
Young leaf tissues
60%
•Sterilizing Jatropha seeds entailed
immersion of seeds in 5% calcium
hypochlorite for 10 min.
•For tissues from existing stocks a
combination of sterilants – 2% Manzate
for 30 min and 5% calcium hypochlorite
for 20 min was used.
•Younger leaf tissues were sterilized for
shorter duration, 20 min., than stem
sections (30 min).
C. Accomplishments and Major
Findings
2. Identification of the best/most
responsive tissue/explant for culture
All tissues from seedlings were
responsive to tissue culture and
more responsive compared to
tissues collected from adult or
mature plants.
Callus readily formed in all types
of tissues
Tissues from adult plants
Cultures from mature/adult plants formed shoots 2 ½
months after inoculation, tissues from seedlings 1 month
after.
J2
Tissues from young plants/seedlings
Jatropha PGL ‘07
1
J10
J 1
J133
leaf tissues
10
J13S2
J02
stem tissues
T2J13S1
stem tissues
root tissues
Different tissues of Jatropha can initiate
shoots
Jatropha
PGL ‘07
J29S2
T2J13S1
stem tissues
Different tissues of Jatropha initiating shoots
#14
J13S2
root tissues
Nov 06 ‘07
Jatropha PGL ‘07
The most responsive explant to shoot
formation is the leaf tissue followed by
stem explants (Table 2).
Table 2. Number of explants developing shoots in
the different culture media tested.
Culture
Medium
M8
M8s
M18
M18ac
M20
M20ac
M22
M22ac
TOTAL
Leaf
explnt
8
1
17
1
6
2
1
2
38
Stem
explnt
1
1
3
2
3
2
1
13
Shoot
tip
1
1
2
1
5
Root
explnt
TOTAL
1
11
3
3
1
8
3
25
3
12
4
4
2
64
• Two types of shoot formation,
direct shoot development and the
development of “embryonic shoot”
were observed
• The leaf cultures had the most
number of direct shoot development
and embryonic shoot formation
compared to stem, shoot and root
cultures (Table 3).
• The direct shoot development
appeared to be the more common
route to shoot formation than the
embryonic shoot.
Callus from leaf of mature plant Direct shoot developing
forming “embryonic” shoot
from callus of leaf tissue
Embryonic shoot formed in Jatropha culture (left)
Jatropha PGL ‘07
Table 3. Type of shoots formed in different culture
media by different explants
Embryonic shoot
Direct shoot
development
TOTAL
Culture
leaf stem shoot root leaf stem shoot root
Mdium
M8
1
M8s
M18
4
M18ac
2
1
1
1
M20
1
M20ac
1
M22
1
1
1
2
22
2
12
3
2
2
2
2
1
3
1
1
1
2
5
1
2
43
9
6
1
4
2
7
34
3
4
M22ac
TOTAL
8
2
6
5
4
73
C. Accomplishments and Major
Findings
3.1 Appropriate medium for callus
formation
Callus formed in all culture
media tested.
Growth and development of callus varied depending upon
J7
the culture media
1-week old
M15
M18
Callus morphology differed
M15
M18
M20
2-week old
M20
Jatropha PGL ‘07
White cottony callus formed in M28ac
Callus in M22 produced shoots.
Embryonic shoots were produced
compared to direct shoot
development in the other media
Nodular calli (left) differentiated to shoots
weeks after (right)
Jatropha PGL ‘07
Different tissues of Jatropha initiating shoots from callus
Jatropha PGL ‘07
C. Accomplishments and Major
Findings
3.2 Appropriate medium for shoot
induction and growth
Different Modified Murashige and Skoog
media (Lapitan 1988) with varying
concentrations and combinations of IAA,
IBA, NAA, Kinetin, BAP and gibberellins
were effective for different developmental
changes in Jatropha tissues cultured.
T2J40 Cultures are more responsive to media without
than with activated charcoal (AC)
M22 w/ AC
M22 w/o AC
J29S2
M18 w/ AC
M18 w/o AC
T2J32
Change in auxin induced the development of new axillary shoot in
less than one week
Jatropha PGL ‘07
#14
Oct 31 ‘07
Jatropha PGL ‘07
The culture media M8, M18 and M20
induced shoot formation better than
the rest of the media tested, with
M18 registering the highest number
of cultures forming shoots followed
by M8 and M20 (Table 2).
Table 2. Number of explants developing shoots in
the different culture media tested.
Culture
Medium
M8
M8s
M18
M18ac
M20
M20ac
M22
M22ac
TOTAL
Leaf
explnt
Stem
explnt
Shoot
tip
Root
explnt
TOTAL
8
1
1
1
11
1
1
1
-
3
17
3
2
3
25
1
2
-
-
3
6
3
-
3
12
2
2
-
-
4
1
1
1
1
4
2
-
-
-
2
38
13
5
8
64
J102
J6
J13S22
J2
Different tissues of Jatropha initiating shoots in M18
Jatropha PGL ‘07
Shoot tips of seedlings growing faster in M8
(left) than in M18
Increasing sucrose of culture
medium induced even more
shoots to form in the cultures.
Gibberellins (medium M7) enhanced and
improved shoot growth. Shoots big enough
for rooting just after 2 weeks.
Protocol for shoot proliferation of mature
tissues has also been established.
Leaf and nodal
tissues were
induced to
develop
multishoots in
media M8 and
M18.
C. Accomplishments and Major
Findings
3.3 Appropriate medium for rooting
Rooting is enhanced in media with
activated charcoal
T2J13S1
A rooted leaf
protocol for rooting still has to
be improved. Outplanting trial
result was not that encouraging.
Survival was only 30%.
C. Accomplishments and Major
Findings
4. Incubation or culture conditions
(light, temperature, photoperiod) for
growth and development of Jatropha
in culture
absence of light can cause the
browning of cultures. Shoot
elongation appeared not
enhanced by short-term
exposure to dark treatment.
Acknowledgement
University of the Philippines Los Banos (Basic Research/Trust
Fund)
ICRISAT, India; UPLB-CHED Jatropha Research Project; homegrown plantations in Los Banos and Batangas.
Chancellor Luis Rey I. Velasco who encouraged the
researcher to conduct this kind of study in support of the
Biofuel Act of the Philippines and UPLB;
Vice Chancellor Enrico P. Supangco who looked for funds
for the project to push through;
Dr. Arturo S.A. Castillo who opened his Jatropha collections
as source of materials for the project;
Ms. Melecia C. Gibe the laboratory technician of the project.