tissue culture of jatropha curcas

Tissue Culture of

Jatropha curcas

Portia Gamboa – LapitanProfessor 7

Department of Forest Biological Sciences

College of Forestry and Natural Resources

10-month Project Report (Aug. 2007 – Jan.

2008, May 1, 2008 to August 31, 2008 )

To develop tissue culture protocol for

the mass propagation of high oil-

yielding varieties of Jatropha

adaptable to the Philippines.

• sterilization scheme for tissues

for culture

• best/most responsive

tissue/explant for culture

A. Project Objectives

• 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

Jatropha PGL ‘07

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

Mature plants of Jatropha for explants

collection were also selected

Jatropha PGL ‘07

Selected plants are sectioned and cultured in

different culture media for organogenesis/plantlet

development

Activated charcoal in the culture medium

facilitates germination of Jatropha seeds

Jatropha Tissue Culture

C. Accomplishments and Major

Findings

1. Sterilization scheme for Jatropha

Sterilization Scheme

Total No. of

Cultures

Ave. No. of Cultures

Contaminated

Ave. Percent

Contamination

Fungal Bacterial Fungal Bacterial

5% calcium hypochlorite for 10 minutes

• Seeds 20/trial of 10

trials

2/trial 1/trial 10% 5%

2% Manzate for 30 min


Leaf Tissues 10/trial of 10

trials

5-6/trial 1/trial 50-60% 10%

Nodal sections 5/trial of 10 trials 3/trial 60%

2% Manzate for 20 min


Young leaf tissues 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 60%

Table 1. Efficacy of sterilization schemes used for Jatropha

tissue cultures

•For tissues from existing stocks a

combination of sterilants – 2% Manzate

for 30 min and 5% calcium hypochlorite

for 20 min was used.

•Sterilizing Jatropha seeds entailed

immersion of seeds in 5% calcium

hypochlorite for 10 min.

•Younger leaf tissues were sterilized for

shorter duration, 20 min., than stem

sections (30 min).

2. Identification of the best/most

responsive tissue/explant for culture


Findings

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

Jatropha PGL ‘07

J2

Cultures from mature/adult plants formed shoots 2 ½

months after inoculation, tissues from seedlings 1 month

after.

Tissues from adult plants

Tissues from young plants/seedlings

Jatropha PGL ‘07

J02

J101

J133

stem tissues

leaf tissues

T2J13S1

J101

stem tissues

Different tissues of Jatropha can initiate shoots

J13S2

root tissues

Nov 06 ‘07 Jatropha PGL ‘07

T2J13S1

#14

J29S2

Different tissues of Jatropha initiating shootsstem tissues

root tissues

J13S2

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

Leaf

explnt

Stem

explnt

Shoot

tip

Root

explnt

TOTAL

M8 8 1 1 1 11

M8s 1 1 1 - 3

M18 17 3 2 3 25

M18ac 1 2 - - 3

M20 6 3 - 3 12

M20ac 2 2 - - 4

M22 1 1 1 1 4

M22ac 2 - - - 2

TOTAL 38 13 5 8 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.

Jatropha PGL ‘07

Callus from leaf of mature plant

forming “embryonic” shoot Direct shoot developing

from callus of leaf tissue

Embryonic shoot formed in Jatropha culture (left)

Table 3. Type of shoots formed in different culture

media by different explants

Embryonic shoot Direct shoot

development TOTAL

Culture

Mdium

leaf stem shoot root leaf stem shoot root

M8 1 8 1 2 12

M8s 1 2 3

M18 4 2 1 1 22 2 2 34

M18ac 1 2 3

M20 1 4 2 2 9

M20ac 1 1 3 1 6

M22 1 1 1 1 4

M22ac 2 2

TOTAL7 5 1 2 43 6 5 4 73


Findings

3.1 Appropriate medium for callus

formation

Callus formed in all culture

media tested.

Jatropha PGL ‘07

Growth and development of callus varied depending upon

the culture media

M15

M15

M18 M20

M20M18

J7

Callus morphology differed

1-week old

2-week old

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


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.

Cultures are more responsive to media without

than with activated charcoal (AC)

M22 w/ AC M22 w/o AC M18 w/o ACM18 w/ AC

Change in auxin induced the development of new axillary shoot in

less than one week

T2J32J29S2

T2J40

Jatropha PGL ‘07

Oct 31 ‘07

Jatropha PGL ‘07

#14

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

Stem

explnt

Shoot

tip

Root

explnt

TOTAL

M8 8 1 1 1 11

M8s1 1 1 - 3

M18 17 3 2 3 25

M18ac1 2 - - 3

M20 6 3 - 3 12

M20ac2 2 - - 4

M22 1 1 1 1 4

M22ac2 - - - 2

TOTAL 38 13 5 8 64

Different tissues of Jatropha initiating shoots in M18Jatropha PGL ‘07

J2J13S22

J102J6

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.


Findings

3.3 Appropriate medium for rooting

T2J13S1

A rooted leaf

Rooting is enhanced in media with

activated charcoal

protocol for rooting still has to

be improved. Outplanting trial

result was not that encouraging.

Survival was only 30%.


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; home-

grown 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.

tissue culture of jatropha curcas

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