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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
Fungal

Contaminated
Bacterial Fungal

Contamination
Bacterial

5% calcium hypochlorite for 10 minutes
• Seeds 20/trial of 10 trials 2/trial 1/trial 10% 5%

2% Manzate for 30 min 5% calcium hypochlorite for 20 minutes Leaf Tissues Nodal sections 10/trial of 10 trials 5/trial of 10 trials 5-6/trial 3/trial 1/trial 50-60% 60% 10%

2% Manzate for 20 min 5% calcium hypochlorite for 10 minutes
Young leaf tissues Shoot tip Young nodal sections 10/trial of 10 trials 5/trial of 10 trials 5/trial of 10 trials 6-7/trial 2/trial 3/trial 1/trial 1/trial 60-70% 40% 60% 10% 20%

•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 PGL ‘07 Jatropha

J133

J101 1 J
10

leaf tissues
J02

J13S2

stem tissues stem tissues
T2J13S1

root tissues

Different tissues of Jatropha can initiate shoots‘07 Jatropha PGL

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 Stem explnt 1 1 3 2 3 2 1 13 Shoot tip 1 1 2 1 5 Root explnt
1

TOTAL
11

38

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 M8s 1 8 1 1 2 2 12 3

M18
M18ac M20 M20ac M22 M22ac
TOTAL

4
1 1 1

2
1 1 1 5

1

1

22
2 4 3 1 2 2 1

2

2
2

34
3 9 6 4 2

1 6 5 4

7

1

2

43

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

M20
2-week old

Callus morphology differed

M15

M18

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
T2J32

M18 w/o AC

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 Leaf explnt
8 1

Stem explnt
1 1

Shoot tip
1 1

Root explnt
1 -

TOTAL
11 3

M8 M8s M18 M18ac M20 M20ac M22 M22ac
TOTAL

17
1

3
2

2
-

3
-

25
3

6
2 1 2 38

3
2 1 13

1 5

3
1 8

12
4 4 2 64

J6

J102

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.