Introduction

Thesis submitted by Mrs. Eram Fatima 1

INTRODUCTION

Energy Independence is one of the vital areas to make India a developed

nation. Among different types of energy sources bio-energy through plant/animal

route has to play a great role as the end of fossil fuel age has already began. Among

the bio-energy, plant route is considered very promising because of its renewable

nature. Plants yielding oil are considered suitable for production of bio-fuel

particularly biodiesel. Because of heavy requirement of edible oil, non-edible oil

yielding plants are considered ideal for Indian condition for production of biodiesel.

Planners and scientists are rightly focusing on non-edible oil yielding plant, Jatropha

curcas, the Physic nut (Singh, 2006).

Jatropha curcas is a plant, which has come in prominence in India in past

couple of years. Earlier forest and agriculture scientists were researching it upon in a

routine manner. Jatropha was mainly known in India among herbalists for its several

medicinal properties. This is a unique plant owned by everyone in Agriculture, Rural

Development, Forest and Environment, Petroleum Non-Conventional Energy,

Panchayati Raj and Finance Ministries. It is not a food, fodder, forest, fibre but a fuel

crop. On fuel crops in our country there are very few research institutes. With the

result improved and high yielding varieties of Jatropha for different regions in the

country are not available. Region specific agro technology packages for Jatropha

cultivation are not ready. Elite planting material, nurseries and certified seeds etc. are

not there. Extraction and esterification of oil from Jatropha seed have lot many

hassles. Continuous supply of seeds throughout the year seems to be a problem due to

which there is low biodiesel production in the country. Price of seed offered to the

farmers and diesel price to the industrialists do not seem to be based on authentic data.

There are several other gaps in the venture requiring immediate attention. In view of

above and other prevailing Jatropha biofuels needs a lots of attention for its growth

and production. At present public and private sectors are promoting Jatropha

production, processing and marketing.

1.JATROPHA

Physic nut (Jatropha curcas L.) is a common plant on the island, sporadically.

The perennial shrub, which can be found in most tropical and subtropical countries all

around the world belongs to the family of Euphorbiaceae and can grow up to 5 meters

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Thesis submitted by Mrs. Eram Fatima 2

height. It has low requirements to soil quality and can grow under low rainfall

conditions the seeds contain about 30 % of uneatable oil (Heller, 1996). In a low input

system the seed yield is about 0.5 to 1t/ha, in a high input system it can reach up to 4

or 5t/ha (Rangan, 2006). As the plant can make use of degraded land, which cannot be

used for growing food, it does not necessarily compete with food production. On the

contrary, as food security not only means ensuring the availability of food, but also

the availability of cooking fuel, the use of locally produced physic nut oil can highly

contribute to food security.

The genus Jatropha of Euphorbiaceae family is one of the prospective

biodiesel yielding tree crops. It is morphologically a diverse genus comprising 160-

175 species of shrubs, rhizomatous shrubs, herbs and small trees. About nine species

of Jatropha have been recorded in India. Out of these important ones are Jatropha

curcas, Jatropha gossypifolia, Jatropha glandulifera, Jatropha multifida, and

Jatropha podagrica. Out of these nine species Jatropha curcas is one of the most

important biodiesel yielding crop. Jatropha curcas commonly called as ratanjyot,

chandrajyot, Jamal gota, Jangli arandi, Kala aranda and physic nut etc, is

multipurpose tree of significant economic importance. It is native of Mexico and

tropical South America (Jubera et. al., 2009). The plant is reported to have been

introduced in Asia and Africa by Portuguese as an oil yielding plant. Now it is

occurring throughout India including Andaman Island in semi wild condition. It is

found throughout most tropics and is known nearly by 200 different names indicating

its significance and various possible uses. It adapts well to semiarid marginal site,

waste land and dry environment. As Jatropha curcas seeds and green leaves are

poisonous that works as a very effective barrier. Long qualified as an interesting but

"underutilized" crop, it is now being increasingly used in reforestation programs in

tropical countries because it thrives on poor soils and on land that is suffering under

erosion (Giovanni, 2007). The use of fossil fuel is also polluting the environment with

carbon dioxide (CO2) which in turn causing global warming. Therefore seed oil from

Jatropha curcas offers an excellent alternative for the source of energy (Jatropha

World, 2007).

1.1 Classification of Jatropha curcas:

Jatropha curcas is a species of flowering plant in the spurge family,

Euphorbiaceae, that is native to the American tropics, most likely Mexico and Central

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America. It is cultivated in tropical and subtropical regions around the world,

becoming naturalized in some areas. The specific epithet, "curcas", was first used by

Portuguese doctor Garcia de Orta more than 400 years ago and is of uncertain origin.

Common names include Barbados Nut, Purging Nut, Physic Nut, or JCL

(abbreviation of Jatropha curcas Linnaeus).

1.2 Scientific Classification:

Kingdom: Plantae

Order: Malpighiales

Family: Euphorbiaceae

Genus: Jatropha

Species: J. curcas

Binomial name: Jatropha curcas L.

1.3 Names in Indian languages:

Bengali: danti, dantigaacha, kochagach

Hindi: danti

Kannada: damti, kaadu haralu, naagadamti

Konkani: baktumbo

Malayalam: ceriyadanthi, naagadanthi

Marathi: danti, katari

Nepalese: ajaya pal, dudhe jhaar

Persian: bedanjire khatai

Sanskrit: anukula, danti, dantika, dirgha, erandhapatrika, erandhaphala,

makulakah,

Tamil: pey-amanakku

1.4 Botanical features:

Leaves: large green to pale-green leaves.

Flowers: male and female flowers are produced on the same inflorescence,

averaging 20 male flowers to each female flower, or 10 male flowers to each

female flower.

Fruits : fruits are produced in winter, or there may be several crops during the

year if soil moisture is good and temperatures are sufficiently high.

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Seeds: the seeds are mature when the capsule changes from green to yellow.

1.5 Importance of Jatropha:

Jatropha curcas L. grows as a large shrub or small tree. The plant is found in

the tropics and subtropics where is used to reclaim land and produce feedstuff, soap,

candle, pesticide and anti-cancer medicine (Openshaw, 2000; Li, et.al., 2001; Lin, et.

al., 2004; Mampane, et. al., 2006).

J. curcas is an antifeedant agent (Meshram et.al., 1994; Adebowale &

Adedire, 2006), a little known herbal drug in dental complaints (Girach et.al., 1995)

and its milky sap is used in Mesoamerica for the treatment of different dermato-

mucosal diseases (Maroquin et.al., 1997). Extracts from the plant are also known for

their medicinal properties and their effects on a wide array of organisms including

insect pests, molluscuns and nematodes (Jain et.al., 1997). It is a multipurpose tree

species fit for agro-forestry and other afforestation programmes (Wood et.al., 1991).

1.6 Utilization of Jatropha as Biofuels production:

Due to the increasing demand for energy and declining fossil fuel resources

(Becker & Francis, 2003) alternative sources of energy are more and more in the

centre of attention. biofuels are such sources of renewable energy. The land which

biofuels are grown on is increasing rapidly in the last years (Zeller & Grass, 2007).

The seed oil ‘Jatropha oil’ can be easily processed to partially or fully replace

petroleum based diesel fuel (Forson, 2004). Thus, the use of this plant for large-scale

bio-diesel production is of great interest with regards to solving the energy shortage,

reducing carbon emission and increasing the income of farmers (Keith, 2000; Zhou

et.al., 2006).

Jatropha seeds contain 46–58% of oil on kernel weight and 30–40% on seed

weight (Subramanian et. al., 2005). It shows promise for use as an oil crop for

biodiesel (Foidl & Elder, 1997; Henning, 1998). The oil is renewable resource and a

safe source of energy and a viable alternative to diesel, kerosene, LPG, furnace oil,

coal and fuel wood (Chandhari & Joshi, 1999). The main advantages of using

biodiesel are its renewability, better quality exhaust gas emission and

biodegradability. It does not contribute to a rise in the level of carbon dioxide in the

atmosphere (Korbitz, 1999; Beet et.al., 2002; Sims, 2001). The oil is also a rich

source of hydrocarbon (27.0–48.5% of seed oil) and in the recent past, Jatropha

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curcas has evoked much interest all over the world as potential petro crop (Martin &

Mayeux, 1985). Such a multiple utility biofuel crop needs genetic improvement in

order to alter its status of wild perennial form to a cultivable crop with higher yield

and oil content.

1.7 Other uses:

Leaves: The young leaves may be safely eaten, steamed or stewed. Cooked with

goat meat, they are said to advantageously counteract its smell. Pounded leaves

are applied near horses' eyes to repel flies in India. HCN (Hydrogen cyanide) is

present in the leaves. The extracts of the plants are dangerous to use but water can

easily release it over if not too much extract is applied.

Flowers: The species is listed as a honey plant. HCN is present.

Nuts: Sometimes roasted and eaten, although they are purgative. They can be

burned like candlenuts when strung on grass. HCN is present. Used as a

contraceptive in South Sudan.

Seeds: Also used as a contraceptive in South Sudan. The oil has been used for

illumination, soap, candles, the adulteration of olive oil, and making Turkey red

oil. Turkey red oil, also called sulphonated (or sulfated) castor oil, is the only oil

that completely disperses in water. It is made by adding sulfuric acid to pure

Jatropha oil. It was the first synthetic detergent after ordinary soap, as this allows

easy use for making bath oil products. It is used in formulating lubricants,

softeners, and dyeing assistants. The seeds in the zone around Misantla, Veracruz

are very appreciated by the population as food once they have been boiled and

roasted. It is unclear if this is due to the existence of a non-toxic variety of

Jatropha in Mexico and Central America, or if the seeds become edible once

processed by cooking. It is also similarly reported that Jatropha seeds are edible

once the embryo has been removed. Again it may be so because of these seeds

coming from a local non-toxic variety. HCN is present.

Roots: Their ashes are used as a salt substitute. HCN and Rotenone are present.

Bark: Used as a fish poison. HCN is present.

Latex: Strongly inhibits the watermelon mosaic virus.

Sap: It stains linen. Sometimes used for marking.

Shrub: Mexicans grow the shrub as a host for the lac insect, which is used in

medicine as hepatoprotective and antiobesity drug.

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1.8 Properties of Jatropha:

Properties Jatropha Oil Jatropha biodiesel Diesel

Density, g/ml 0.920 0.865 0.841

Viscosity @ 400C 3.5 5.2 4.5

Calorific value, MJ/kg 39.7 39.2 42.0

Flash point, 0C 240 175 50

Cloud point, 0C 16 13 9

1.9 Process for biodiesel production and by-products from Jatropha seeds:

Raw Jatropha oil will cause carbon deposits on the piston and head of an

engine due to high viscosity of oil, which in turn results in incomplete fuel

combustion. The above problem can be solved by transesterification of raw oil to give

biodiesel. The biodiesel pilot plant consists of a transesterification reactor with a

heater, a stirrer, a chemical mixing tank, three glycerol settling tanks and a washing

tank. The capacity of the pilot biodiesel plant is 250 litres/day. The cost of the pilot

plant is Rs. 2.5 lakhs. The process flowchart for biodiesel production and pilot

biodiesel plant are shown in Figures below:

Process flowchart for biodiesel production from Jatropha seeds and byproducts

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1.10. Indian Scenario of Jatropha:

Former President Kalam discussed that this alternative fuel source could

transform India's oil scenario with Jatropha-growing farmers in Sundarkera village,

located on the outskirts of Chhattisgarh state capital Raipur and said India plans to

produce 60 million tonnes per annum of biofuel by 2030. Speaking to the farmers cultivating

Jatropha at mass level, he said: Jatropha is a vital tree for bio-diesel. Farmers should use only the

high oil-content quality saplings and they must do trimming at the right time in the first year to

split the tree into at least 60 branches, so that a single tree can produce 400 grams of seeds in a

year. Wishing good luck to the farmers to bring in a biodiesel revolution in the country, Kalam

added that the government and private sector major players should accelerate research in the

biofuel sector, as well as the related aspects of production, marketing and processing

In other states, the oil content in Jatropha seeds is below 25 %, but in

Chhattisgarh the oil content percentage is over 30, Kalam said in response to a query

raised by a farmer. He advised them to use only high oil content quality seeds and

proper irrigation can further increase oil yields. The president said that the

Government would take care of the financial aspects of Jatropha growers, including

proper marketing and aid in Jatropha irrigation.

Chief Minister Dr. Raman Singh announced immediately that he would

provide 75% subsidy to farmers for Jatropha irrigation through drip and sprinkler

systems. The Chhattisgarh Government claims that biofuel rich plants like Jatropha

and Karanj have the potential to help India get over its annual requirement of 124

million metric tonnes of petroleum products, of which around 72% is met through

imports at a cost of more than Rs. 1.5 trillion. It has announced plans to plant 160

million saplings across the state in the current fiscal year. It proposed to run all state-

owned vehicles with Jatropha fuel instead of imported diesel by 2007.

India is not sufficient in edible oils; non-edible oil is the main choice for

producing biodiesel. Some development works have been carried out with regards to

the production of transesterfied non-edible oil and its use in biodiesel by units such as

the Indian Institute of Science, Bangalore, Tamil Nadu Agriculture University

Coimbatore and Kumaraguru College of Technology, in association with Pan Horti

Consultants in Coimbatore, Tamil Nadu. Generally, a blend of 5% to 20% is used in

India (B5 to B20). The Indian Oil Corporation has taken up research and development

work to establish the parameters of the production of transesterfied Jatropha vegetable

oil and use of biodiesel in its R & D centre at Faridabad. Research is carried out at the

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Thesis submitted by Mrs. Eram Fatima 8

Kumaraguru College of Technology for marginally altering the engine parameters to

suit the Indian Jatropha seeds and to minimise the cost of transesterification.

Recently, the State Bank of India provided a further boost to the cultivation of

Jatropha by signing a memorandum to give to local farmers in India, for the harvested

Jatropha seeds. The Indian Railways have started to use Jatropha oil blended with

diesel to power its diesel engines with great success. Many Indian states have already

jumped onto the Jatropha train, including Andhra Pradesh, Chhattisgarh, Karnataka,

Tamil Nadu, Rajasthan and Maharashtra.

Jatropha has been held up as a reliable source of income for India’s poor rural

farmers, providing energy self-sufficiency, while reducing fossil fuel consumption

and greenhouse gas emissions. Several states have distributed plants free of charge to

small farmers, encouraging private investment in Jatropha plantations and setting up

biodiesel processing plants. The Ministry of Rural Development, which is to

coordinate the national mission on biofuel when it is approved, estimates that there

are already between 500,000 to 600,000 ha of Jatropha growing across India.

Chhattisgarh has the well-developed Jatropha biodiesel programme in the

country. It has given away 380 million Jatropha seedlings to farmers, enough to cover

150,000 hectares, and also provided 80 oil presses to various village governing bodies

with guarantees to buy back Jatropha seeds at Rs. 6.5 per kg. Several local micro-

refinery businesses have sprung up across the state to provide biodiesel for tractors,

irrigation pumps, jeeps and village power generators. The widespread government

support has attracted foreign investments. UK-based D1 Oils, the world’s largest

commercial cultivator of Jatropha, has around 80,000 ha in Chhattisgarh and in the

southern state of Tamil Nadu, with plans for an additional 350,000 ha over the next

several years. The state Government funds Jatropha seeds and D1 Oils guarantees to

buy the harvested seeds at the price prescribed by the state.

D1 Oils’ Indian operations is focusing on research on yield, and the company

is testing a number of Jatropha varieties to find which grows best in India’s varied

climatic regions. Former President Dr Abdul Kalam is a strong advocate of Jatropha

biodiesel. In a speech in 2006, he said ...India has a potential to produce nearly 60 million

tonnes of biofuel annually using 30 million hectares of land, thus making a significant and

important contribution to the goal of Energy Independence. Indian Railways has already

taken a significant step of running two passenger locomotives (Thanjavur to Nagore

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section) and six trains of diesel multiple units (Tiruchirapalli to Lalgudi, Dindigul and

Karur sections) with a 5% blend of biofuel sourced from its in-house esterification

plants. In addition, they have planted 75 lakhs Jatropha saplings on Railway land,

which is expected to give yields from the current year onwards. Similarly some of the

States, such as Chhattisgarh, Andhra Pradesh, Madhya Pradesh, Uttaranchal and

Tamil Nadu have energy plantations in India

Need For In Vitro Regeneration:

In order to meet the demand of fuel in the near future, the development of appropriate

technology for the rapid regeneration of this species is essential. The conventional

method of propagation through seeds will not solve the problem. Under this situation

in vitro regeneration of this species through tissue culture techniques offers a

powerful method to overcome the problem. Intervention of biotechnological methods

to introduce desirable traits in Jatropha species is the need of the hour. Tissue-culture

protocols for endosperm cultures and the rapid propagation of selected genotypes of

Jatropha have been reported by various researchers (Spera, et.al., 1997).

2.Tissue Culture Techniques:

Tissue culture is a well established technology for the ornamentals and some

horticultural species. Micropropagation, popularly known for large-scale clonal

propagation, is the first major and widely accepted practical application of plant

biotechnology. Now it has gained the status of a multibillion dollar industry

throughout the world. Initially, the technique of micropropagation for large-scale

production of plants was employed basically to ornamental plants only (Ammirato et.

al., 1989), but recently it has been extended to various vegetable and fruit crops such

as potato, strawberry, oil palm, banana, etc. medicinal and aromatic plants and trees

(Bajaj et al, 1986, 1988). Presently, the micropropagation technique is especially

being used not only for those plants which are difficult to be propagated through

conventional practices, but also for the mass multiplication of existing stocks of

germplasm for biomass energy production and conservation of important, elite and

rare plant species that are threatened or on the verge of extinction (Wawrosch, et. al.,

2001; Dhar, et. al., 2000).

During the past couple of decades, there has been an increased interest in

problems related to the large-scale plant production as well as in its cost reduction for

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commercial micropropagation (Donnan, 1986; Chu & Kurtz, 1989; Andrea-Kodym &

Zapata-Arias, 2001). Somatic embryogenesis i.e., the formation of embryo like

structures from somatic cells and their germination into the complete plants

(Bhaskaran & Smith, 1990) has now been proved as potentially most efficient and

economic method for the large-scale clonal propagation of plants. The potential of

somatic embryogenesis for unlimited multiplication of plants gives tremendous

advantage to micropropagation technology. The plants developed from somatic

embryos are normally true to type or less variable as compared to the plants

developed via shoot and root morphogenesis.

2.1 Commercial Aspects of Micropropagation:

In last few years, tissue culture has emerged as a commercially viable venture

for the developing countries. These laboratories, however, are thriving on the exports.

Tissue culture propagation is labour intensive and wage rates being high in developed

countries, it is expected that the demand of tissue culture propagated plants will

remain stable in the future. It is estimated that the market for tissue culture plants is

enormous and up to ten times the present production level can be accommodated in

the international market.

3. Use of Molecular Marker:

Jatropha species are essentially cross pollinated, which result in a high degree

of variation and offers the breeder ample scope to undertake screening and selection

of seed sources for the desired traits (Ginwal, et. al., 2005). Selection is the most

important activity in all tree breeding programmes (Zobel & Talbert 1984). Since

variability is a prerequisite for selection programme, it is necessary to detect and

document the amount of variation existing within and between populations. DNA

marker based fingerprinting can distinguish species rapidly using small amounts of

DNA and therefore can assist to deduce reliable information on their phylogenetic

relationships. DNA markers are not typically influenced by environmental conditions

and therefore can be used to help describe patterns of genetic variation among plant

populations and to identify duplicated accessions within germplasm collections.

Various approaches are available for DNA fingerprinting such as amplified fragment

length polymorphism (AFLP) (Zabeau & Vos 1993), restriction fragment length

polymorphism (RFLP) (Botstein, et. al.,1980), simple sequence repeats (SSRs)

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(Tautz,1989) and randomly amplified polymorphic DNAs (RAPD) (Williams, et.

al.,1990). Among these, RAPD is an inexpensive and rapid method not requiring any

information regarding the genome of the plant, and has been widely used to ascertain

the genetic diversity in several plants (Belaj, et. al., 2001). RAPD analysis requires

only a small amount of genomic DNA and can produce high levels of polymorphism

and may facilitate more effective diversity analysis in plants (Williams, et. al., 1990).

Since variability is a prerequisite for selection programme, it is necessary to

detect and document the amount of variation existing within and between populations.

DNA marker based fingerprinting can distinguish species rapidly using small amounts

of DNA and therefore can assist to deduce reliable information on their phylogenetic

relationships. DNA markers are not typically influenced by environmental conditions

and therefore can be used to describe patterns of genetic variation among plant

populations and to identify duplicated accessions within germplasm collections.

Various approaches are available for DNA fingerprinting such as amplified fragment

length polymorphism (AFLP), restriction fragment length polymorphism (RFLP),

simple sequence repeats (SSRs) and randomly amplified polymorphic DNA (RAPD).

Among these, RAPD is an inexpensive and rapid method not requiring any

information regarding the genome of the plant, and has been widely used to ascertain

the genetic diversity in several plants. RAPD analysis requires only small amount of

genomic DNA and can produce high levels of polymorphism and may facilitate more

effective diversity analysis in plants and it provides information that can help to

define the distinctiveness of species and phylogenetic relationships at molecular level.

Use of such techniques for germplasm characterization may facilitate the conservation

and utilization of plant genetic resources, permitting the identification of unique

genotypes or sources of genetically diverse genotypes (Ganesh, et. al., 2007).

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Thesis submitted by Mrs. Eram Fatima 12

Inflorescence

FRUITS

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Thesis submitted by Mrs. Eram Fatima 13

Objectives:

The objectives of the present study were as follows:

Selection of different population of Jatropha curcas from different area of

Chhattisgarh region.

To develop high quality of planting material in nursery through seed

germination of Jatropha curcas.

Initiation of culture through different type of explant from seedling and mature

plant of Jatropha curcas.

To study the influence of different media (MS, SH, WPM and B5), PGR,

provenance and adjuvant (s) on shoot proliferation of seedling and mature

plant of Jatropha curcas.

Subculture of micro-shoots and determination of shoot multiplication rate in

seedling and mature plant of Jatropha curcas.

To study the effect of IBA, IAA and NAA on in vitro rooting of micro-shoots

of seedling and mature plant of Jatropha curcas.

Acclimatization of in vitro raised plantlets derived from seedling and mature

plant.

To study the somatic embryogenesis/callus and shoot initiation.

DNA extraction in Jatropha curcas from different provenancenes.

To study the genetic diversity in Jatropha curcas population by RAPD

analysis.