Energy Procedia 47 ( 2014 ) 21 – 28

1876-6102 © 2014 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Scientifi c Committee of Indonesia EBTKE Conex 2013doi: 10.1016/j.egypro.2014.01.192

ScienceDirect

Conference and Exhibition Indonesia Renewable Energy & Energy Conservation [Indonesia EBTKE CONEX 2013]

Somatic Embryogenesis Media Optimization Study of Physic Nut (Jatropha curcas) as Biodiesel Feedstock

Anggi Ninditaa,*, Bambang Sapta Purwokoa, Darda Efendia, Iswari S. Dewib aDepartment of Agronomy and Horticulture, Bogor Agricultural University, Jl. Meranti, Kampus IPB Darmaga,

Bogor 16680, Indonesia bIndonesia Center for Agricultural Biotechnology and Genetic Resources Research and Development, Ministry of Agriculture,

Indonesia, Jl. Tentara Pelajar No. 3A, Kampus Penelitian Pertanian Cimanggu, Bogor 16111, Jawa Barat, Indonesia __________________________________________________________________________________________________________________________________________

Abstract

Jatropha curcas as potential biodiesel feedstock is difficult to propagate through tissue culture. The research development for Jatropha is massive nowadays and propagation can be conducted through conventional or non-conventional techniques in biotechnology. Biotechnology approach through organogenesis and embryogenesis pathways is needed. The objectives of the research were to obtain in vitro culture media optimization through embryogenesis pathway. Plant materials used in this experiment were embryo axis and cotyledon to obtain somatic embryo. The result of the experiment showed that somatic embryos were obtained only from MS medium supplemented with picloram 1.0 mg/l for both embryo axis and cotyledon explant. © 2014 The Authors.Published by Elsevier Ltd. Selection and peer-review under responsibility of the Scientific Committee of Indonesia EBTKE Conex 2013.

Keywords: physic nut; in vitro; picloram; embryo axis; cotyledon _____________________________________________________________________________________________ 1. Introduction

Availability of energy source especially fossil fuel becomes the main problem in the world and also particularly in Indonesia. Biofuels as a part of a portfolio of solutions to high energy prices, including conservation, more efficient energy use, and use of other alternative fuels. Biofuel production is expected to increase as new plants being built begin production. Indonesia government need to look for alternative energy and encourage biofuel _________________________________ * Corresponding author. Tel.: +6281317723381 E-mail address: angginindita@yahoo.com

Available online at www.sciencedirect.com

© 2014 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Scientific Committee of Indonesia EBTKE Conex 2013

22 Anggi Nindita et al. / Energy Procedia 47 ( 2014 ) 21 – 28

industry due to energy based fossil oil crisis. Jatropha curcas as one of potential biodiesel feedstock can be alternative for this problem solve. Jatropha has its own advantages, relatively easy to cultivate in field by farmers, can grow in marginal lands and efficient processing of castor oil [1]. Global environmental problems, such as climate change and global warming support the biofuel development. Biofuel development expected can absorb employment, reduce unemployment, and increase the income through its cultivation. Biofuel development expected can utilize agro industry and its byproduct.

Jatropha curcas is claim as rich secondary metabolites species. It is also valuable multi purposes crops. Jatropha seeds produce oil, with simple refinery the oil is ready to be produce into biodiesel and burn oil [2]. It is important to obtain and evaluate best genotype for Jatropha oil contents and quality. [2-3]. Genetic diversity in Jatropha and genetic transformation is necessary to observed.

Propagation study through embryogenesis pathway was conducted to support material supply and meet demand of fuel in the near future [3]. Unsuccessful Jatropha conventional methods of propagation through seeds [4] utilize tissue culture [5] techniques to overcome supply plant materials. Recent study had been conducted to established jatropha tissue culture protocol [6-7] and its propagation study. Variation type of explant will give suboptimum result in tissue culture regeneration [8]. Plant growth hormones (i.e. auxin and cytokinin) are commonly used for tissue culture regeneration. Persistence of auxin and cytokinin ratio can induce callus and organogenesis [9]. Micropropagation of Jatropha integerrima [10, 11, 12] are reported to develop Jatropha research. Multiplication of explant by using embryogenesis somatic is one of technique that can be applied and develop [13]. With this technique explant can be induced to form somatic seedling from several specific stages: embryogenic callus induction, maturation, germination, and hardening. The objectives of the research were to obtain in vitro culture media optimization through embryogenesis pathway.

2. Materials and methods

2.1. Plant material

Material used are Jatropha improve population named IP-1P. Jatropha IP-1P was collected and evaluated accessions in Indonesia from Dompu, Lampung, Sumba, Cigawir, Cibedug and Banten. 2.2. Explant sterilization

The seed were peeled and soaked in sterile water under room temperature. Seed were sterilized using 20% sodium hypochlorite solution from 20 minutes. The seed then rinsed five times with sterilized water. Inside the laminar flow, the seed were dried and cut. Embryo axis and cotyledone were extracted for embryogenesis induction. 2.3. Media optimization

Optimization for callus induction was conducted to obtain media combination treatment. The experimental design was randomized complete block design (RCBD) with two factors. Treatments implemented with 20 repetitions. Each repetition used 5explant. First factor is type of explant i.e. embryo axis and cotyledone. Types of auxin as treatment from 2.4-D and Picloram for induced embryogenic callus were used as second factor (Table 1). The explants were planted 45 days with no light incubation. Callus induction and embryogenic callus was counted. Table1. Combination treatment from type of auxin and type of explant and explants.

Type of Explant

Type of Auxin

Code of Control 2,4-D (mg/l) Picloram (mg/l)

1.0 2.0 3.0 5.0 3.0 1.0

Embryo axis M1 M2 M3 M4 M5 M6 M7

Cotyledon M8 M9 M10 M11 M12 M13 M14

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2.4. Observation

Explant growth and development was observed each week. Variables measured i.e. percentage of explants forming callus, development of callus, color of callus from explant, percentage of calli forming somatic embryos, number of somatic embryos and day of somatic embryos emerge.

Fig. 1. Scoring of development callus: (A) 1 – 25 % Callus(scor value 2), (B) 26 – 50% callus coverage explant (scor value 3), (C) 51 – 75 % callus coverage explant (scor value 4), (D) 76 – 100 % callus coverage explant (scor value 5).

Fig. 2. Scoring of Callus color: (A) 76 – 100 % brown callus (score value 1), (B) 21 – 75 % brown callus (score value 2), (C) no color callus (score value 3), (D) Green callus (score value 4).

2.5. Statistical analysis

Analysis of variance and mean comparison computed by statistical analysis system (SAS). Duncan range test is used to analyze significant different.

3. Result and discussion

3.1. Experiment condition

At the beginning of this experiment, the development of callus from explants did not show significantly different results between treatments. At two weeks after transplanting explants usually just swell. Explants were used in this experiment is the explants derived from Jatropha seeds. Explant type used is the embryonic axis and cotyledons

A

C

B

D

A

C

B

D

24 Anggi Nindita et al. / Energy Procedia 47 ( 2014 ) 21 – 28

embryos derived from Jatropha seeds. Table 2 shows the percentage number of explants forming callus and somatic embryo explants number. The

concentration of plant growth regulators 2,4-D and Picloram can induce the formation of callus on explants of embryonic axis and cotyledons up to 100% at 12 WAP (week after planting). Response on each explant callus formation is different in response to the formation of somatic embryos. The concentration of plant growth regulators picloram 1.0 mg / l can induce somatic embryo formation by 65% in explants derived from embryonic axis and 45% in explants derived from the cotyledons. Embryonic axis and cotyledon explants can be used as a source of explants for callus formation in vitro, but the success of callus formation cannot be used as an indication of success in the process of formation of somatic embryos, callus formed because not necessarily embryogenic callus. Table 2. Effect of type of auxin and eksplant to percentage of explants forming callus and somatic embryo.

Type of Auxin explants forming callus (%) explants forming Somatic Embryo (%)

Embryo axis Cotyledon Embryo axis Cotyledon

Control 100 0 0 0

2.4-D 1.0 mg/l 100 100 0 0

2.4-D 2.0 mg/l 90 100 0 0

2.4-D 3.0 mg/l 100 90 0 0

Picloram 1.0 mg/l 100 100 65 45

Picloram 3.0 mg/l 100 100 0 0

Picloram 5.0 mg/l 100 100 0 0

3.2. Development and color of callus

Results of analysis of variance elucidated an interaction between the level of concentration of growth regulators

to the type of explant on callus growth is not real and the real color of the callus. Scores callus development of the

highest type of explants derived from embryonic axis explants type that is equal to 4.0 (Table 3). Tabel 3. Effect from type of explant to callus development score.

Type of explant Callus development score

Embryo axis 4.0 a

Cotyledon 3.3 b

Note: Mean followed by the same letter are not significantly different based on the DMRT test at 0.05 level.

Results of analysis of variance showed an interaction between the level of concentration of growth regulators to the type of explant on callus growth is not real and the real color of the callus. Scores callus development of the highest type of explants derived from embryonic axis explants type that is equal to 4.0 (Table 3).

Anggi Nindita et al. / Energy Procedia 47 ( 2014 ) 21 – 28 25

Tabel 4. Score of callus development.

Type of Auxin Score of development callus

control 1.2d 2.4-D 1.0 mg/l 3.7 b 2.4-D 2.0 mg/l 3.5 bc 2.4-D 3.0 mg/l 3.5 bc

Picloram 1.0 mg/l 4.6 a Picloram 3.0 mg/l 4.5 a Picloram 5.0 mg/l 4.6 a

Note: Mean followed by the same letter are not significantly different based on the DMRT test at 0.05 level.

Table 5 shows the treatment picloram 1.0 and 3.0 gives a score of more than 4 colors callus on explants of embryonic axis and cotyledon explants. Score the highest callus color was on MS (Murashige & Skoog) medium + Picloram 1.0 mg / l. The callus color score was 4.8. It is suspected that the embryogenic callus produced. Figure 3 shows the alleged embryogenic callus forming globular stage. [14] states that the non-embryogenic callus formation in explants inhibits excessive formation of somatic embryos and regeneration of arabica coffee. Tabel 5. Effect of treatment to color of callus.

Type of Auxin Color of callus score

Embryo axis Cotyledon Cotyledon 1.0 e 1.0 e

2.4-D 1.0 mg/l 4.0 b 4.0 b 2.4-D 2.0 mg/l 3.0 c 3.0 c 2.4-D 3.0 mg/l 3.0 c 3.6 c

Picloram 1.0 mg/l 4.8 a 4.8 a Picloram 3.0 mg/l 4.0 b 4.0 b Picloram 5.0 mg/l 3.0 c 3.6 c

Note: Mean followed by the same letter are not significantly different based on the DMRT test at 0.05 level.

Fig. 3. Embryogenic callus in globular stage.

26 Anggi Nindita et al. / Energy Procedia 47 ( 2014 ) 21 – 28

3.3 Number of somatic embryo

The results showed that there was an interaction between the concentration of growth regulators and explant type

on the number of somatic embryos at 12 WAP. Although some callus on the color scores high treatment (> 4), but

apparently not all of embryogenic callus. Somatic embryos formed in the embryo axis explants in media containing

Picloram 1.0 mg / l only in the amount of 5.1 to 1.1 and embryo axis explants in explants derived from cotyledons

(Table 6).

Tabel 6. Effect of treatment to number of somatic embryo.

Type of Auxin Number of Somatic Embryo

Embryo axis Cotyledon

Control 0 b 0 b

2.4-D 1.0 mg/l 0 b 0 b

2.4-D 2.0 mg/l 0 b 0 b

2.4-D 3.0 mg/l 0 b 0 b

Picloram 1.0 mg/l 5.1 a 1.1 a Picloram 3.0 mg/l 0 b 0 b

Picloram 5.0 mg/l 0 b 0 b Note: Mean followed by the same letter are not significantly different based on the DMRT test at 0.05 level.

Results of research conducted by [12] and [15] showed that BAP (benzyl amino purine) and kinetin were able to induce somatic embryos. [12] obtain direct somatic embryos, which plays an important role BAP induce somatic embryogenesis from cotyledon explants of Jatropha, while [15] suggested that kinetin 2.0 mg / l are better able to induce embryogenic callus from young leaf explants compared with BAP.

In this experiment the compact callus formed in the dark, while the friable callus formed a bright place. Compact callus formed allegedly due to the influence of Picloram concentration. Muchtar stated that the problems encountered in the use of plant growth regulators is a careful selection of the appropriate type and concentration of the physiology of plants and explants were grown [16]. Selection of the type and concentration of the difference is due to the type of plants and plant tissue has a specific response to the administration of growth regulators. Explants forming somatic embryos at the globular stage, torpedo and cotyledon is shown in Figure 4.

[16] showed that the formation of somatic embryos on rattan manauauxin maximum occurs at the level of 2 ppm Picloram, culture with concentration of growth regulator 2,4-D up to 4 ppm is not well formed embryos combined with BAP and kinetin. [17] showed that the sandalwood somatic embryos can be formed directly by using growth regulators BAP up to 4 ppm.

Fig. 4. Somatic embryo form (A) globular, (B) torpedo, and (C) cotyledon stage.

Anggi Nindita et al. / Energy Procedia 47 ( 2014 ) 21 – 28 27

3.4. Embryo somatic emergence

Figure 5 shows somatic embryo observed under microscope. Somatic embryo forms a bipolar structure, which at

bud part has primary leaf and apical dome.

Fig. 5. Microscopic observation of somatic embryo with stereo microscope.

Picloram medium with MS + 1.0 mg / l of somatic embryos raises an average of 30 days after planting (DAP). Picloram is an auxin type whose main function is to stimulate cell elongation [18]. Use of Picloram 3.0 mg / l and 5.0 mg / l in this study turned out to cause browning (browning) on callus so Picloram at high concentrations can’t be used to induce somatic embryos. Something similar happened to treatment with concentrations of growth regulators 2,4-D. Picloram and 2,4-D in addition to functioning as an auxin also serves as a herbicide.

Conclusion

The result of the experiment showed that somatic embryos were obtained only from MS medium supplemented with picloram 1.0 mg/l for both embryo axis and cotyledon explant.

Acknowledgement

We thank the IPB its staffs for their helpful assistance in conducting the field experiment. Generous funding support from the Directorate General of Higher Education, Ministry of Education, Indonesia are highly appreciated.

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