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6

American-Eurasian Journal of Sustainable Agriculture, 6(1): 6-13, 2012


ISSN 1995-0748

ORIGINAL ARTICLE
Progress In Jatropha Curcas Tissue Culture
1
Ahmed Saad Attaya, 1Danny Geelen and 2Abd El-Fatah Helmy Belal
1
Dept. of Plant Production, Bioscience Engineering Faculty, Ghent University, Belgium.
2
Dept. of Plant Production, Environmental Agricultural Sciences Faculty, Suez Canal University, Egypt.

Ahmed Saad Attaya, Danny Geelen, Abd El-Fatah Helmy Belal; Progress In Jatropha Curcas Tissue
Culture

ABSTRACT

The last decade witnessed a blooming interest in the development of in vitro culturing technology for the
energy crop Jatropha curcas. As a result, a series of papers were published reporting on callus induction,
somatic embryogenesis and micropropagation. This study aim to give an overview about reliable and highly
efficient tissue culture protocols for direct and callus mediated shoot regeneration and somatic embryogenesis
are established for J. curcas which indicates potential for widening the genetic base through biotechnological
tools. A variety of explants have been used that produced callus in the presence of different auxins and
cytokinins and conclude that J. curcas gave better growth of callus in medium with high concentrations of
auxin and cytokinin. Shoot tips have been used to propagate Jatropha successfully, however, the quality of the
shoots and their capacity to regenerate roots are not yet sufficiently robust to allow commercial implementation.
We also point out the biotechnological advantages of J. curcas tissue culture and indicate that accessions used
for developing propagation protocols were mainly originating from Asia where the genetic diversity of the J.
curcas is limited. Further optimization of micropropagation conditions and the implementation of American
accessions will be required to develop commercially viable in vitro propagation methods for J. curcas.

Key words: Jatropha curcas plant tissue culture micropropagation in vitro rooting organogenesis explant.

Introduction

J. curcas (L.) the common name physic nut, known as Purgative nut, Barbados nut, Retanjot etc., belongs to
the family Euphorbiaceae. Many of the family members have some ornamental use except J. curcas and J.
glandulifera that are oil-yielding species. J. curcas is considered as a potential crop for local fuel production. At
optimal conditions, about 5 tons/ha seed is produced per year. The hull and the kernel have a high oil content
that can reach up to about 60% and which can be transformed into fuel through esterification (Li et al., 2007).
The oil is high in cetane value and can be used directly in diesel engines added to diesel fuel as an extender or
transesterized to a biodiesel fuel. Due to the presence of several toxins including curcascine or curcin, phorbol
esters, saponins, protease inhibitors and phytates, the seed or the oil cannot be used for human or animal
consumption (Data et al., 2007; Joubert et al., 1984; Makkar et al., 1998; Menezes et al., 2006; Munch and
Kielfer, 1989). In contrast to the varieties used for oil production, some Mexican J. curcas accessions are edible
(Makkar et al., 1998). In addition, Jatropha oil is used in making soaps, candles, paints, lubricants and medicine
(Sujatha and Mukta, 1996).
The latex of J. curcas contains an alkaloid known as "jatrophine" which is believed to have medicinal
properties. The sap of the plant is used for treatment of piles, snakebite, skin diseases, paralysis, dropsy, malarial
fever, arthritis, gout, jaundice and resistance to various stresses (Heller, 1996; Openshaw, 2000).
Because of this variety of Jatropha products, it is cultivated by peasant farmers. It is desired because of its
rapid growth, easy propagation, low cost of seeds, high oil content, small gestation period, wide adaptation,
production on good and degraded soils and the optimum plant size that makes the seed collection convenient
(Francis, 2005; Jones and Miller, 1991; Kumar and Sharma, 2008). J. curcas can also be used for reclamation
of waste lands (Achten et al., 2007).
Perhaps one of the most important properties of Jatropha is that it survives short periods of drought and
therefore can be used in more arid regions where other oil crops like palm cannot grow. Moreover, some of the
semi-arid land areas are subject to erosion (Abdulla et al., 2011). The root architecture of J. curcas involves a
pen root that grows to great depths highly suitable for fixing the soil and the prevention of erosion (Openshaw,
2000).
Corresponding Author: Ahmed Saad Attaya, Dept. of Plant Production, Bioscience Engineering Faculty, Ghent University,
Belgium.
E-mail: ahmd.attaya@gmail.com

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Am.-Eurasian J. Sustain. Agric. 6(1): 6-13, 2012

Conventional agriculture uses seeds and cuttings for its propagation. J. curcas is self-compatible but tends
to cross-pollination producing seed from about 30% through self-pollination (Qing et al., 2007). Hence many
seed is heterozygous and it is beset with problems of poor seed viability, low germination, scanty and delayed
rooting of seedlings. Vegetative cuttings are seasonal and were reported to generate plants with a lower
longevity, a lower drought and disease resistance, and poor root system lacking a tap root that is required for
strong anchorage (Sujatha et al., 2005). Seed production begins when the plants are 3-4 years although the
production number is still rather small. One year old saplings can be used for cuttings (Jones and Miller, 1991).
J. curcas is becoming a commercial source of biodiesel production in several West-African (Senegal) and
South-East African (Zambia, Tanzania, Namibia) countries, as well as in Asia (Philippines and especially in
India) where state governments are actively promoting its commercialization (Fig 1). Energy experts claim that
Jatropha oil is an environmentally safe, cost-effective renewable source of non-conventional energy, and a
promising substitute for diesel, kerosene and other fuels (Abdulla et al., 2011).
Because of the increased interest in the potential of Jatropha as an energy plant, more attention is given to
methods that allow the mass production of elite material. The method most suitable for generating large
numbers of plantlets is in vitro propagation. Hence the rapid growing list of reports on tissue culture analysis of
J. curcas.

Biotechnological Advantages Of Jatropha Tissue Culture:

Plant cell and tissue culture techniques provide an alternative approach to the plants which are difficult to
cultivate, or a long cultivation period, or has a low yield and have been used for propagation of many plant
species and offer rapid and continuous supply of the planting material (Thepsamran et al., 2006). J. curcas
tissue culture studies have been conducted to develop a fast and efficient multiplication system. (Sujatha et al.,
2005) indicate that in vitro plants of J. curcas produce a better yield and yield-related traits than seed-
propagated plants. However, this system may be preferred above cuttings because of its flexibility to adjust to
the market demand and the production of pathogen free material. Moreover, tissue culture technologies would
help in producing the active compounds in vitro with better productivities without cutting down the natural
resources.
Apart from the benefits of in vitro propagation, regeneration protocols have also been developed with the
aim to genetically transform J. curcas. (Li et al., 2007) established an efficient genetic transformation via
Agrobacterium tumefaciens infection of cotyledons and found about 33% of the resistant calli differentiated into
shoots by using phosphinothricin as a selective agent. (Pan et al., 2010) found that 30.8% of the kanamycin-
resistant regenerated J. curcas plants transformed using polymerase chain reaction (PCR), Southern blot
analysis and -glucuronidase (GUS) activity staining. (Purkayastha et al., 2010) reported a genetic system in J.
curcas using bombardment of particles coated with plasmid pB1426 containing a GUS-NPT II reporter. These
methods will be important for generating transgenic material with improved agriculturally important properties
such as timing and abundance of female flower development, and resistance to pests.
(Divakara et al., 2010) indicated that transgenic approach helps in genetic modification and subsequent in
vitro multiplication for various uses and to improve seed productivity and oil biosynthesis pathway of J. curcas
which requires an efficient genetic transformation. Genetic engineering through transformation has been widely
used to obtain transgenic plants through some stages in molecular and cellular biological techniques is another
valuable method for the development of J. curcas. A new full length cDNA encoding aquaporin (JcPIP2) was
isolated from J. curcas seedlings the abundance of JcPIP2 was induced by heavy drought stress and it plays an
important role in rapid growth of Jatropha under dry conditions (Ying et al., 2007). Moreover, a new full length
cDNA of stearoyl-acyl carrier protein desaturase was obtained by RT-PCR and RACE techniques from
developing seeds of Jatropha and the gene was functionally expressed in Escherichia coli (Tong et al., 2006).
(Zhang et al., 2008) established a novel betaine aldehyde dehydrogenase gene (BADH) named JcBD1 expressed
in leaves undergoing environmental stress like drought (30% PEG), heat (50C) and salt (300 mM NaCl).
Another investigation by (Tang et al., 2007) about JcERF gene showing enhanced resistance to salt and frost.
Jatropha tissue culture has enabled (Qing et al., 2007) to study haploid culture and self pollinating out
crossing, the results of breeding system indicated 32.9% fruit setting under self pollination. The fruit sets of
artificial self-pollinated was 87.9%, which indicated that J. curcas was self compatible and tended to cross-
pollination. Also, it has enabled (Kaewpoo and Te-chato, 2009) to study polyploidization, they confirmed that
there is no variation in ploidy level of micropropagated plants derived from both epicotyls and hypocotyls
culture.

J. Curcas Accessions Used For Tissue Culture:

The origin or accession of a plant that is used for in vitro cultivation is a critical factor that will determine
the cultivation requirements and the protocol needed for a successful in vitro culturing. That is why it is

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Am.-Eurasian J. Sustain. Agric. 6(1): 6-13, 2012

important to indicate the source of the material when publishing a tissue culture method. Jatropha grows in
tropical to subtropical climates and is therefore mainly occurring around the equator. It is still uncertain where
the centre of origin is but based on the highest level of genetic divergence detected in Mexico it is believed that
Jatropha originated somewhere in Central America (Belize, Costa Rica, El Salvador, Guatemala, Honduras,
Nicaragua and Panama). (Aponte, 1978) reported that J. curcas is most likely native to Central America as well
as to Mexico where it occurs in the forests of coastal regions. Natural populations of Jatropha occur throughout
the Neo tropic from Mexico to Brazil including the Caribbean Island. Subsequent to the discovery of the
Jatropha tree by the Portuguese, it was distributed via the Cape Verde Islands and Guinea Bissau (Heller, 1996).
Nowadays J. curcas is cultivated world-wide including Central and South Africa, India and the Pacific regions
of Asia.

Fig. 1: World map of Jatropha research and cultivation activity. The brown dots indicate the countreis where J.
curcas is cultivated. The blue square dots point out the regions where J. curcas accession were taken for
tissue culture research: India (North Guwahat, Assam; Chorvadla; Bhopal; Bavistin; Andhra Pradesh;
South of Andhra Pradesh; Rajasthan; Kolkata; Jaipur; Lucknow; Coimbatore), Pakistan (Islamabad),
China (Guizhou province, P.R.), Thailand (Bangkok), Malaysia, Brazil and Mexico.

Germplasm conservation centers are located in three countries (in CATIE, Costa Rica, 12 provenances in
CNSF, Burkina Faso and one provenance in INIDA, Cape Verde). Most of the tissue culture investigations were
done with seeds from different locations in India followed by much fewer studies in Pakistan, China, Thailand,
Malaysia, Brazil and Mexico. Some research is also conducted in Indonesia and South Africa, however so far no
publications have been released from these countries. Clearly, the majority of in vitro propagation studies were
done with J. curcas accessions from the regions with the least biological diversity. It is therefore pertinent that
additional studies should be conducted to analyze the behavior and properties of cultivars coming from the
Americas.

Tissue Culture Techniques Implemented For Jatropha Curcas:

In Vitro Seed Germination:

Surface sterilization is essential prior to in vitro incubation to avoid microbial contamination. The most
effective methods of sterilization involve the decoating of the seed and subsequent soaking in tap or distilled
water for 24-48 h (Deore and Johnson, 2008 ; Soomro and Memon, 2007) at room temperature. In a following
step the seeds are surface sterilized with commercial detergent or 70% (v/v) ethanol and immersed in 0.1 %
(w/v) mercuric chloride solution followed by sterile distilled water rinsing (wash three to four times) to remove
the traces of HgCl solution (Li et al., 2007; Shrivastava and Banerjee, 2009; Soomro and Memon, 2007). The
surface sterilized seed germinated on MS medium.

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Explants Used For The Initiation Of Tissue Cultures:

A wide range of explants have been used for the introduction of J. curcas cultures: leaf, petiole, stem,
cotyledons, epicotyls, hypocotyls, penduncle, nodal segments, apical shoots and axillary bud-derived shoots
(Kaewpoo and Te-chato, 2009; Rajore et al., 2002; Sardana et al., 1998; Sujatha and Mukta, 1996; Sujatha and
Reddy, 2000; Thepsamran et al., 2008; Wei et al., 2004). The use of cotyledons, epicotyls and hypocotyls
generates clones from individual seedlings and hence most propagules are genetically distinct given that the
seeds derived mainly from outcrossing events. These explants are therefore not suitable for the production of
elite clones.

Induction of Callus:

A variety of tissue has been used for the induction of callus. Both the application of auxin as well as high
cytokinin concentrations were effective. Here we give an overview of the explants and conditions that have been
reported. Callus produced with 0.5 mg l-1 2,4-D using hypocotyls explants grew fast during first 7 to 30 days of
culture and then stabilized at a slower growth rate in the medium (Monacelli et al., 1995). The addition of 2%
v/v coconut milk to this medium was shown to have a similar effect as without the coconut supplement
(Hoshino et al., 1995; Kawak et al., 1995; Soomro and Memon, 2007). The application of BA (0.5 mg l-1) alone
or in combination with IBA (0.1 mg l-1) was also very effective in inducing callus (Sujatha and Mukta, 1996).
When TDZ was used at 1-3 mg/l, compact white and green callus was formed without the regeneration of
shoots.
When leaf explant was used, high concentrations of BA (5.0 mg l-1) with NAA (1.0 mg l-1) induced callus
within 3-4 weeks (Rajore and Batra, 2007). Interestingly, NAA (1.0-4.0 mg l-1) alone in MS also induce
callus. This finding suggests that J. curcas is highly sensitive to auxin with respect to the stimulation of cell
division rather than the induction of roots (Shrivastava and Banerjee, 2008). Replacing NAA by IBA in
combination with BA did induce callus (Deore and Johnson, 2008; Sujatha and Mukta, 1996; Thepsamran et al.,
2008). However, IBA on its own was not sufficient to stimulate callus formation. More complex hormone
mixtures were used by (Soomro and Memon, 2007) who treated leaf explants from 4 days old seedlings with
different growth regulators 2,4-D, BA, GA3 and coconut milk.
Finally, embryo explants have been used for the induction of callus with the aim to generate embryonic
callus that can be used for somatic embryogenesis. Here, MS medium was supplemented with a very high
amount of NAA (1.25 mg l-1) and Zeatin (60 g l-1) (Astha et al., 2006). The callus obtained was green, friable
and embryogenic.
In conclusion, in terms of number of callus and hormone reactivity, epicotyls and hypocotyls explants and
zygotic embryos were the most suitable explants material. Auxin and cytokinin can independently induce callus
with BA and NAA as the most potent hormones. These findings indicated that J. curcas responds to high auxin
and high cytokinin primarily by stimulating cell division.

Micropropagation Methods Developed For Jatropha Curcas:

A. Cotyledon Culture:

Cotyledons of J. curcas are easy to collect from germinating seed. They are about 5-10 mm in length and
remain attached to the seedling during development. Cotyledons were the preferred explants for transformation
of J. curcas and were more susceptible to Agrobacterium infection than other explants such as petioles,
hypocotyls, epicotyls or leaves (Li et al., 2006). Many adventitious buds are formed on the margins or the inner
regions of cotyledon explants after 3 weeks incubation on medium containing BA (1.5 mg l-1) and IBA (0.05 mg
l-1). 33 to 38% of the total inoculated explants produced adventitious shoots. Although cotyledons are the
preferred explants by Li et al. it should be noticed that this method is not suitable for elite plant propagation as
the seedlings are genotypically dissimilar.

B. Nodal Stem Segment Culture:

By and large the most frequently used explants for the induction of shoot tip cultures is nodal stem
segments. Nodal meristems are an important tissue source for micropropagation and plants raised from these are
comparatively much less prone to genetic variation (Pierik, 1991). Shoot bud proliferation from axillary nodes
was assessed on MS basal salt medium supplemented with different kinds of cytokinin. (Sujatha and Reddy,
1998) showed that TDZ promoted higher shoot regeneration frequency from axillaries as compared to BA. In a
later study, the concentrations (0.5-10 mg l-1) from each Kn, BA and TDZ were tested, followed by subculture
to medium with 2 mg l-1BA (Sujatha et al., 2005). The shoot proliferation rate was about 10 fold for nodes

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cultured on medium containing 0.5 and 1 mg l-1 TDZ. The presence of TDZ has a greater influence on the
induction of adventitious shoot buds than other cytokinins (Deore and Johnson, 2008). Yet, with BA a similar
multiplication factor was obtained. (Shrivastava and Banerjee, 2008) propagated about 10 shoots/axillary node
on MS with 3.0 mg l-1 BA, 1.0 mg l-1 IBA, 25 mg l-1 adenine sulphate, 50 mg l-1 Glutamine, 15 mg l-1 L-arginine
and 25 mg l-1 Citric acid within 3-4 weeks, and (Singh et al., 2009) produced 10 -15 shoots/axillary node on MS
medium supplemented with 1.0 mg l-1 BA in combination with 1.0 mg l-1 Kn. On high cytokinin medium, the
morphological characteristics of the propagated shoots were not optimal. Therefore, (Kaewpoo and Te-chato,
2009) used lower cytokinin levels 0.5 mg l-1 BA and 0.25 mg l-1 IBA to induce adventitious shoots directly from
stem, axillary bud and shoot tip explants achieving a multiplication factor of about 5 shoots/explants. (Rajore
and Batra, 2007) reported optimal conditions as 1.5 mg l-1 BA and 0.5 mg l-1 IBA.
(Thepsamran et al., 2008) demonstrated shoot regeneration from various types of explants from apical
shoots, nodes, axillary bud-derived shoots, petioles and leaf explants on MS medium supplemented with
different concentrations of BA alone or in combination with IBA. MS supplemented with 0.5-1 mg l-1 BA was
effective for apical shoot culture 1 mg l-1 BA was suitable for node culture, while 0.5 mg l-1 BA in combination
with 0.01 mg l-1 IBA provided the best shoot proliferation from axillary bud-derived shoots.

C. Leaf and Petiole Culture:

Several authors have reported direct shoot bud formation from leaves and petioles on MS medium in
presence of hormones. (Deore and Johnson, 2008; Khurana-Kaul et al., 2010; Kumar and Reddy, 2010; Soomro
and Memon, 2007; Sujatha and Mukta, 1996; Sujatha et al., 2005; Thepsamran et al., 2008). A major advantage
of using leaf explants is that it has greater multiplication potential. This is because the nodal segment
propagation is limited to the number of axillary buds whereas leaf explants may induce a multitude of new
shoots, depending on the regeneration capacity. The downside of the use of leaf explants is that a shorter or
longer episode of callus formation precedes the initiation of a shoot. Hence, callus derived somaclonal variations
may turn up in the propagated material.
Leaf discs show a good response to BA (2 mg l-1) and IBA (0.5 mg l-1) as 80% to 90% of the explants
produce adventitious shoots (Sujatha et al., 2005). Leaf explants cultured on MS medium supplemented with 0.2
mg l-1 TDZ in combination with 0.2 mg l-1 IBA produced adventitious shoot buds (18.8 0.6) directly without
formation of intervening callus. thus, proliferation of shoots improved when they were transferred to PM2
medium containing CuSO4 concentrations, maximum shoot proliferation (22.8 0.8) was obtained when shoot
buds were induced and subcultured on medium with 0.15 mg l-1 CuSO4 (Khurana-Kaul et al., 2010).
(Thepsamran et al., 2008) the maximum percentage of explants producing shoots was 70%, while the maximum
number of regenerated shoots was 5.4 obtained from petiole segments of leaves at the second node of branches.
Leaf discs from the third expanding leaf exhibited higher regeneration potential than those from the fourth
leaf suggesting that the developmental stage of the Jatropha is an important determinant in hormone responses
(Sujatha and Mukta, 1996). It also suggested that plant juvenility is a relevant factor in the capacity to
regenerate. Older leaf explants also show regenerative capacity but these require different hormone mixtures. In
particular TDZ and a high cytokinin concentration is required in these instances (Deore and Johnson, 2008).
(Kumar and Reddy, 2010) developed an efficient and reproducible protocol for the regeneration of J. curcas
from petiole explants without formation of an intervening callus using MS medium supplemented with 0.5 mg l-
1
TDZ. They reported 58.3% shoot bud induction and 10 shoots per explant were obtained.

Root Regeneration:

The induction of rooting by auxins is commonly applied to regenerate viable plants from shoots and
cuttings (Gunes, 2000). IBA was shown to be effective in the rooting of J. curcas (Kochhar et al., 2005). Other
studies confirm the root induction capacity of IBA albeit at different concentrations and with different
efficiencies. IBA induced roots in 52% of the shoots within three weeks in medium containing 0.1-0.2 mg l-1
IBA (Datta et al., 2007). In 40% rooting was achieved with 0.1 mg l-1 IBA after 5 weeks (Singh et al., 2009) and
in 85.71% rooting induced from shoots on MS basal medium with 1 mg l-1 IBA (Thepsamran et al., 2008).
(Kaewpoo and Techato, 2009) reported a high rate of root induction frequency of 60% was obtained after 30-40
days on MS medium supplemented with 0.5 mg l-1 IBA. (Deore and Johnson, 2008) reported well-developed
shoots were rooted on MS medium supplemented with 0.1 mg l-1 IBA after 30 days. Rooting was comparatively
better in half strength MS medium (Shrivastava and Banerjee, 2008; Rajore and Batra, 2005; Khurana-Kaul et
al., 2010).Also microshoots rooted well on MS + IBA (3.0 mg l-1) and the plantlets successfully acclimatized in
soil (Rajore and Batra, 2007).
Rooting was effectively achieved on full strength MS supplemented with 1.0 mg l-1 IAA within 6-7 days
(Kalimuthu et al., 2007). However, (Kumar and Reddy, 2010) maintained that strength MS medium

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supplemented with 2% sucrose, 3 mg l-1 IBA in combination with 1 mg l-1 IAA, 1 mg l-1 NAA and 0.25 mg l-1
activated charcoal found to be the best for promoting rooting.
In general, rooting was achieved in the presence of IBA whereas IAA and NAA were less effective and
often induce the formation of callus at the base of the shoot.

Somatic Embryogenesis And Embryo Culture:

(Astha et al., 2006) developed an regeneration protocol from embryo cultures. Somatic embryos were
cultured on MS medium supplemented with different concentrations of NAA, IAA, IBA and 2,4-D in
combination with Zeatin. Induction of globular somatic embryos in 58% of the incubated cultures was achieved
on MS basal medium supplemented with different concentrations of Kn in combination with IBA. In a separate
study 0.5 mg l-1 Kn + 0.2 mg l-1 IBA proved to be the most effective for somatic embryo induction (80%) in J.
curcas (Jha et al., 2007). (Kalimuthu et al., 2007) showed somatic embryo induction directly from green
cotyledon explants on MS supplemented with 2 mg l-1 of BA. Recently, (Shrivastava and Banerjee, 2009)
showed direct organogenesis from J. curcas embryo cultures.

Conclusion:

A series of studies show that J. curcas is amenable for propagation in vitro. However, it seems that the
results of the different reports vary despite the fact that mostly the same hormone analogs were applied. It is also
surprising that most of these studies were conducted in Asia where the J. curcas genetic variability is rather
small, suggesting that the variations observed are largely due to cultivation conditions. Although that some
successes were made, it is clear that the rooting of in vitro J. curcas shoots is not fully optimized and will
require further research. In addition, it will be important to test a wider range of genetically different accessions
in order assess the impact of the genotype on tissue culture conditions. Finally, J. curcas transformation
protocols have been developed starting from seedling tissue. For commercial application it will be important to
develop a protocol that can be applied to clonal tissue.

Abbreviations:

BA Benzyladenine
Kn kinetin
TDZ Thidiazuron
IBA Indole-3-butyric acid
NAA -Naphthaleneacetic acid
IAA Indole-3-acetic acid
MS Murashige and Skoog medium

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