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Procedia Chemistry 14 (2015) 239 – 245

2nd Humboldt Kolleg in conjunction with International Conference on Natural Sciences,

HK-ICONS 2014

In Vitro Selection of Jatropha curcas Linn. Hybrids Using

Polyethylene Glycol to Obtain Drought Tolerance Character
Maftuchaha*, Agus Zainudina
a
Laboratory of Vegetable Oil Technology, Agriculture Faculty, University of Muhammadiyah Malang,
Jl. Raya Tlogomas 246 Malang – 65144, East Java, Indonesia

Abstract

This research aims to repair the character of drought tolerance at F1 hybrids of Jatropha curcas Linn. by in vitro selection
using polyethylene glycol. The seeds of J. curcas genotypes were sterilized before germinated on a sterile sand medium. The
zygotic embryos then grown in MS media + 0.5 mg·L–1 BAP + 0.4 mg·L–1 TDZ. The lethal dossage value was reached 73.57 %
from treatment of 15 % PEG, and 20 % of PEG treatment was reached 91.43 %. In selection media, the percentage of
death explants ranged from 76.00 % to 93.50 %. The callus that obtained, then propagated and developed into regeneration media
and expected could be developed completely plantlets which would have drought tolerance.

© 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
© 2015 Maftuchah, A. Zainudin. Published by Elsevier B.V.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014.
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014
Keywords :Callus; drough tolerant; in vitro selection; Jatropha curcas Linn.; polyethylene glycol (PEG)

Nomenclature
BAP benzyl amino purine
J. curcas Jatropha curcas Linn.
LD lethal dossage
MS murashige skoog
PEG polyethylene glycol
TDZ thidiazurone
wk week

* Corresponding author. Tel.: +62 816 1435 516; fax: +62 341 460 782.
E-mail address: maftuchah_umm@yahoo.com

1876-6196 © 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014
doi:10.1016/j.proche.2015.03.034
240 Maftuchah and Agus Zainudin / Procedia Chemistry 14 (2015) 239 – 245

1. Introduction

The recent problem facing global issues is the increasing of fossil fuels necessity while the supply trend is
declining. This situation has been led to the finding of new or renewable energy sources. It is important as part of the
energy resources diversification program. Biofuel could be optional energy resources in order to substitute fossils
energy. Besides reducing fuels dependency (as an energy diversification), the use of bio-fuel has positive impact on
environment because bio-fuel has low pollution, biodegradable, non-toxic1, and is also able to reduce gas emissions
of green house effect2.
High yield varieties is needed for the development of Jatropha curcas plant as a source of biodiesel. However,
there are not adequate varieties yet 3. Until now, Indonesia has not obtained high yield varieties of Jatropha curcas.
So, the seeds still depend on farmer collections that are dispersed on different regions of Indonesia. The availability
of high genetic variation of J.curcas is an important factor in breeding programs to obtain the new varieties. In
order to increase the genetic variation, various techniques could be done, including through the application of in
vitro technology. In vitro culture of plant cells, tissues or organs on a medium containing selective agents offers the
opportunity to select and regenerate plants with desirable characteristics. The technique has also been effectively
utilized to induce tolerance which includes the use of some selective agents that permit the preferential survival and
growth of desired phenotypes4
The somaclonal method from in vitro technology could affect on the events of genetic variation on regenerated
materials that resulted from spontaneously regenerated of somatic cells. The genetic variations are may be expressed
from morphological level to metabolic production and molecular level5. The variation of somaclonal could be
produced from the effect of genetic variation of explants and/or tissue culture applications. Cells mutation and
polisomic effects in certain tissues of plant may cause the variation of explants. The genetic variation that resulted
from tissue culture may cause by doubling number of chromosomes, chromosomal structure changes, genes and
cytoplasm changes6. So, from in vitro culture could be selected the genotypes that are useful for plant breeding
program.
The somaclonal variation method is commonly used in in vitro techniques. This method is more effective and
efficiently because the changes of traits could be more directed to the expected traits. The changes of genetic traits
will be more efficiently and increasing when into the media is adding by certain organic components that could
affect on genetic variation7. The induction of somaclonal variation that applied on in vitro selection methods has
been used to select the level of drought tolerant on many types of plants through the addition of polyethylene glycol
(PEG) compound. The addition of PEG has been reported to be able to decrease water potential on culture media of
in vitro technique and make it efficiently to obtain the genetic variation of plants which are tolerant to drought
stress8. The use of PEG additions to obtain the genetic variants of plant has been used in rice, sorghum, and grapes 9.
The availability of in vitro techniques which are effectively to regenerate plantlets in a large number of
plants and then followed by producing the somaclonal plants is the one of important factor that may determine the
successful of in vitro selection technique, especially to obtain the somaclonal lines with certain promising traits
which may be expected on target plants. Besides saving of time and spaces, by presence of stress on cells
or tissues in selection media of in vitro which giving in homogeneous condition is expected to be more efficiently in
results5. The in vitro selection technique to get drought tolerant on sugarcane somaclonal variation has been obtained
from the 60 g · L–1 of PEG concentration which was based on the color of callii, compactness of callii, speed of
formation roots of buds, stem height, number of shoots, root number and root length6. In other plants, the using
of PEG concentrations is ranging from 25 g · L–1 to 80 g · L–1 6,9.
The purpose of this study was to improve the trait of drought tolerance from promising clones of
J. curcas based on in vitro selection using polyethylene glycol (PEG).

2. Material and methods

This research was carried out in one year by using seven numbers of Jatropha curcas Linn. crossing. The
materials used were Jatropha curcas10 seeds, Murashige and Skoog culture media, BAP and IBA hormones,
thidiazurone, polyetilene glycol, sterile sand, sterilizing other compounds (tween 20, benlate, 90 % alcohol,
 Maftuchah and Agus Zainudin / Procedia Chemistry 14 (2015) 239 – 245 241

50 % chlorox, and sterile distilled water), in vitro culture equipments, etc. The PEG concentrations that were
applied on the in vitro selection for challenge to drought tolerant clones included 0 %, 5 %, 10 %, 15 %,
20 % and 25 %11,12.

The research activities consist of several stages as follow:

2.1. Initiation and maintenance of embryogenic callus

The seeds of J. curcas genotypes resulted from F1 of crossing plant were sterilized before being germinated
on sterile sand medium, like also other compounds including tween 20, benlate, 90 % alcohol, 50 % clorox and
sterile distilled water. In 2 wk of normal germination, the percentages of young plants viability were observed. The
young plants from Jatropha seeds then used as source of tissue culture materials to produce the zygotic embryos.
The zygotic embryos then grown in induction media for young embryogenic callii that contain of MS media +
0.5 mg · L–1 BAP + 0.4 mg · L–1 TDZ. Each population genotype of Jatropha seeds used 100 zygotic embryos (10
explants per vial) for being cultured by in vitro. After 4 wk to 8 wk of culture period, the percentage of zygotic
embryos that formed callii were observed and also percentage of embryogenic callii formation.

2.2. Polyethylene glycol (PEG) treatment

To evaluate the effect of PEG on the development of embryogenic callii of J. curcas, somatic embryogenic
callii that formed explants at 8 weeks after plantation was cultured on the basis of MS liquid medium by adding
BAP (0.5 mg · L–1) and IBA (0.2 mg · L–1 to 0.4 mg · L–1) hormones12 . Afterward, various treatments of PEG
concentrations (MW 6000) were done by varying different concentration at 0 %, 5 %, 10 %, 15 %, 20 % and
25 %11. The development of embryogenic callii in cultured selection media were observed in 4 wk
after cultured. The observations included the percentage of living callii. Whereas, the sub-lethal of PEG
concentration was determined by the level of PEG giving that inhibit the normal growth of callii at the age of 4 wk
after being cultured.

2.3. In vitro selection on media containing PEG

The sub-lethal concentration of PEG was determined for drought tolerant selection on J.curcas at somatic
embryos phase. The tested embryogenic callii in each population genotype was then selected by culturing these
calli into in vitro media selection which containing of PEG at sub-lethal concentrations level
(obtained from previous stages). Each of F1 population genotype was cultured as much as 200 of explants callii
(10 explant callii/vial). During selection process of in vitro, explant callii were incubated in the dark condition and
placed at room-temperature or about 25 °C r 2 °C of environment temperature. Observations were done at the
number of living callii and also the percentage of living abilities of callii.

2.4. Regeneration of variants somatic embryogenic callii with drought tolerant

The somatic embryogenics callii which were able to survive on selection media with contained of PEG then
was sub-cultured on maturation media (MS + BAP 0.5 mg · L–1) until somatic embryogenics callii were completely
formed.

2.5. Regeneration of plantlets with drought tolerant

After obtained complete somatic embryogenic callii, then regenerated callii on MS basic media that was added
with BAP (0.5 mg · L–1) and IBA (0.2 mg · L–1 to 0.4 mg · L–1) hormones until somatic embryogenic callii
developed into complete plantlets. Cultures were incubated at room-temperature 25 r 2 °C with 1 lm · m2
illumination for 16 h (1 lx equal 1 lm · m2).
242 Maftuchah and Agus Zainudin / Procedia Chemistry 14 (2015) 239 – 245

3. Result and discussion

The selection procedure for drought tolerant on J.curcas was carried out based on in vitro selection method
which was treated by polyethylene glycol (PEG) compound. In order to produce required materials for in vitro
culture, the Jatropha curcas seeds were propagated by planting these seeds in sterile sand media. Afterwards, in the
next step the cotyledon that had been obtained from germinated seeds was isolated and then cotyledon was cultured
in Murashige & Skoog basic media to acquire embryos zygotic callii. Table 1 shows the percentage of normal
germination of Jatropha seeds in sterilized sand media.

Table 1. The percentage of normal seed germinated from Jatropha curcas seeds which planted in sterilized sand media
Number of Number of seeds Number of normal seeds Percentage of normal
Code Notes
seeds germination germinated seeds germinated (%)
5 50 49 48 96.00 1 abnormal
6 50 48 44 88.00 4 abnormal
7 50 45 43 86.00 2 abnormal
18 50 47 46 92.00 1 contaminated
The average of normal seed germinated (%) 90.50

Table 1 shows that the F1 seeds from number five accession plant resulted the highest normal germination
percentage (96.00 %) while the F1 from number seven accession plant seeds produced the lowest percentage
of normal germination (86.00 %). The average percentage of normal germination of four accessions were
90.50 %. Abnormality of sprouts in this study were shown by: hypocotyl small, small cotyledons, and partly not
open cotyledon. Figure 1 shows J. curcas sprouts grown on sterile sand medium.

Fig 1. Jatropha curcas sprouts grown on sterile sand medium

Table 2. The percentage of J.curcas embryogenic callus formation on MS medium + 0.5 mg · L–1BAP + 0.4 mg · L–1 TDZ.
Embryogenic callus Percentage of embryogenic callus
Code Number of embryos zigotic
(8 wk) formed (%)
5 100 76 76.00
6 100 82 82.00
7 100 65 65.00
18 100 69 69.00
Average 73.00

Table 2 shows the percentage of embryogenic callii formation on MS medium with 0.5 mg · L–1 BAP and
0.4 mg · L–1 TDZ. Plant of F1 from six accession were give the highest percentage of embryogenic
callii formation (82.00 %) while the lowest percentage of embryogenic callii formation was seven accession
(65.00 %). In this research, the average percentage of embryogenic callii formation from all populations were
73.00 %.
 Maftuchah and Agus Zainudin / Procedia Chemistry 14 (2015) 239 – 245 243

Table 3. The concentration levels of PEG tested for callii selection on in vitro media of J.curcas plants at 4 wk old
Concentration PEG (%) Number of samples Number of living callii Percentage of living callii (%) LD (%)
0 140 140 100,00 0,00
5 140 118 84.29 15.71
10 140 106 75.71 24.29
15 140 37 26.43 73.57
20 140 12 8.57 91.43
25 140 0 0 100.00

Sub-lethal concentrations of PEG were determined as the concentration of PEG that can inhibit
the normal growth of somatic embryos at 4 wk old. In this study, 15 % PEG concentration was chosen as the
medium selectors because this concentration had the percentage of lives callus of 26.43 % (LD = 73.57 %).
Figure 2 indicates the explants were not able to pass (A) and able to pass (B) the selection medium of PEG.
Callii that passed selection will still fresh and have green color (B), where they generally could be grown and could
develop as healthy plantlets, while callus that did not pass the selection will change their color to
brown and eventually die.
High molecular weight Polyethylene glycol has long been used to stimulate drought stress in plants13. PEG of
high molecular is a non-penetrating osmotic agent lowering the water potential in a way similar to soil drying14. The
ability of PEG to become a negative water potential could be used as an agent to assume of plant tissue response by
drought stress15,16. PEG 6000 is an osmotic agent that could not cause plasmolysis and non toxic for plants16, so the
death of callus in medium with PEG was not caused by absorption of PEG to the cells, but by decreasing water
potential in the medium.

A B

Fig 2. Explants did not pass (A) and passed (B) the medium selection of PEG

Table 4 shows the results of in vitro selection from few number accession of J. curcas that grown on PEG of
15 % concentration. The data also indicates that treatment of 15 % PEG could lead to explants death by
percentage ranged from 76.00 % (five accession) up to 93.50 % (seven accession). The average
percentage of the explants death of fourth plants accession tested were 83.75 %. Figure 4 shows the embryogenic
callus of Jatropha was selected by PEG 15 % at eight weeks old. From selection process by in vitro method which
was treated by 15 % PEG, the callii that were able to pass the PEG selection after 1 mo to 2 mo would
grow into small pieces of shoots. Small shoots then were transferred or sub-cultured into the regeneration
media. Shoots in sub-cultured process aimed to obtain a rather large-sized of individual
shoots. Individual shoots that have a large enough size then transferred to a medium for root formation17. The
J. curcas plantlets then maintained until they formed a complete plant (leaves and stems) and then ready to be
transferred to the acclimation medium. Figure 3 showed the embryogenic callus of J. curcas plant that
was selected by using of PEG 15 % at 8 wk old.
244 Maftuchah and Agus Zainudin / Procedia Chemistry 14 (2015) 239 – 245

Table 4. Selection results from a number of J.curcas by in vitro technique at 15 % of polyethylene glycol (PEG) treatment
Code Number of explants Number of live calli Percentage of death explants (%)
5 200 48 76.00
6 200 32 84.00
7 200 13 93.50
18 200 37 81,50
Average of death explants 83,75

Fig 3. Embryogenic callus of J. curcas that was selected by using of PEG15 % at 8 wk old

Figure 4. J.curcas L. plantlets were resulted from PEG selection in the regeneration media.

Figure 4 shows plantlets of J. curcas that resulted from PEG selection in the regeneration medium. In culture
media there were able to produce plantlets with leaves. Thus, by in vitro selection techniques have been proven that
utilization for the selection process of many kinds of plant species to drought tolerant. The PEG compound has been
reported to be able to decrease water potential of in vitro culture media to get the plant drought - tolerant variant8
and also it has been successfully performed on many species of plants, such as rice, sorghum and grapes5,6. The
availability of an effective technique of in vitro selection model to regenerate plantlets in large number and produce
of somaclonal variant plant population is one of the requirements to the successful application of in vitro
selection to obtain strains of somaclonal variants with promising specific traits. Besides saving time and space, the
presence of stress on cells or tissues in selection media of in vitro that gives homogeneous condition is expected to
give more efficient results5.

4. Conclusion

This research has produced plantlets of J. curcas from selection of in vitro media of which treatment
using PEG. The optimization of plantlets results that application of in vitro selection using PEG showed that
 Maftuchah and Agus Zainudin / Procedia Chemistry 14 (2015) 239 – 245 245

more higher concentrations of PEG given would be followed by increasing the LD value. In the 15%
of PEG treatment, the LD value reached 73.57 %, in 20 % of PEG, LD value reached 91.43 %. While, in selection
media that contained 15 % of PEG, the percentage of death explants ranged from 76.00 % to 93.50 %. The J. curcas
that were resulted from in vitro selection using 15 % PEG have been able to multiply and be developed in the
regeneration media and plantlets which were able to develop became plants that have perfect drought tolerant trait.

Acknowledgements

The gratitude goes to General Directorate of Higher Education and National Department of Education for
funding of this research. The tribute is addressed to the entire staffs of the In Vitro Culture Laboratory, University
of Muhammadiyah Malang for their assistance and equipment provided during the process of the research.

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