PETROTECH 2009 11 - 15 JANUARY 2009, NEW DELHI, INDIA P09-869

BIOTECHNOLOGICAL INTERVENTIONS JATROPHA AND CASTOR FOR BIOFUELS

M. Sujatha
1

FOR

IMPROVING

Directorate of Oilseeds Research, Rajendranagar, Hyderabad 500 030, India E-mail: mulpurisujata@yahoo.com

ABSTRACT
This article highlights the role of biotechnological tools in the genetic improvement of Jatropha and castor belonging to the family euphorbiaceae. Ever increasing fuel prices and depletion of fossil reserves demands alternative fuel sources. For a country like India with predominantly rainfed agriculture, biofuels from water demanding crops and food crops is a difficult proposition and hence, tree borne and non-edible oilseed crops are being researched for exploitation as bioenergy crops. Among the potential biofuel crops, Jatropha and Pongamia assumed priority owing to their easy adaptability, growth on harsh environments and use of transesterified oil in diesel engines. Pongamia is a tree species and improvement through conventional breeding and biotechnological tools is a long drawn process. India holds monopoly in castor area and production and can be exploited for production of biodiesel (www.castoroil.in). While castor is valued for the oil and its derivatives in a host of medicinal and industrial products, the utilitarian value of Jatropha oil and its by-products need extensive investigations. The limitations in available germplasm of Jatropha include; lack of knowledge of the genetic base, poor yields on problematic sites, low genetic diversity and vulnerability to wide array of insects and diseases. During the past three years there is an upsurge of activities in jatropha and information on molecular markers, gene sequences, microsatellite database and cloning and characterization of useful genes (curcin, stearoyl-acyl carrier protein desaturase, JcERF for enhanced resistance to salt and frost) is generated. Tissue culture protocols are refined and regeneration through direct adventitious and somatic embryogenesis is reported. Genetic transformation protocols are in place which indicates potential for widening the genetic base through biotechnological tools. Different types of molecular markers are used in assessment of genetic diversity in the germplasm and should pave way for marker assisted breeding. In castor, the major limitations include susceptibility to biotic stresses and presence of the toxic protein ricin in the seed meal which are being addressed through transgenic approach and TILLING, respectively. Castor genome sequencing has been initiated at the Institute for Genomic Research (TIGR) and around 50,000 expressed sequence tags (ESTs) have been produced for gene identification and annotation. Bioengineering of fatty acid metabolism pathway in both these crops has huge scope for downstream processing and value-chain expansion. The current status and future prospects of these technologies for genetic upgradation of Jatropha and castor will be discussed.

Introduction
India imports petroleum crude supplies to meet 70% of the energy requirement and the energy demand in the country is increasing at a rate of 6% annually, which is a major limitation threatening sustainable development. Of the several options available, biofuels from energy crops has attracted attention as they are renewable when compared to the fossil fuels that are finite, combat climate change, and as an alternative for the ever increasing raw petroleum prices. The production of biofuels has to be considered against different aspects such as energy yield per unit area under cultivation, food versus fuel needs, potential for substitution of fossil fuels, cost of production and competitiveness with fossil fuels, overall energy balance and net reduction potential of greenhouse gas emissions, sustainable agricultural practice, competition with food and animal fodder and energy, trade and agricultural policy issues (1). The developed nations have the option for exploitation of food crops for biofuel purpose, but introduction of first-

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PETROTECH 2009 11 - 15 JANUARY 2009, NEW DELHI, INDIA

generation biofuels has led to substantial increases in raw material prices. Developing nations like India has to rely on non-edible oilseed crops to avoid competition of land for food crops. Globally, more than 300 tree borne oilseeds (TBOs) have been identified as suitable raw materials for biodiesel of which about 100 plant species occur in wild or cultivated condition in India. Preliminary evaluation of several tree oilseed crops for their growth, productivity and adaptability points to identification of plant species such as Jatropha curcas, Pongamia pinnata, Simarouba glauca, Calophyllum inophyllum, Hevea brasiliensis, Madhuca indica, Shorea robusta, Mesua ferra, Mallotus phillippinensis, Garcinia indica, Salvadora, etc., to be promising as per the agro-climatic regions of the country. However, the first choice would be for crops that are suitable for marginal lands. Castor and J. curcas are undemanding non-edible oilseed crops which can thrive well in a variety of climatic, rainfall and soil conditions besi8des being renewable and completely biodegradable. These crops avoid the competition on land use for food and animal fodder. The crops are toxic and not browsed by cattle. In case of J. curcas, all plant parts have utilitarian value viz., young leaves as food; seeds, leaves and bark in traditional medicine and for veterinary purpose; leaves, seed oil and phorbol esters from the oil as plant protectant and molluscicide; seed oil in soap production; seed cake as organic manure; hulls of fruits and seeds as burning material (2). The use of different J. curcas components has been specified (3) but the commercial potential of J. curcas oil and its derivatives need to be established. Till date, there are no authentic records of actual yield potential of J. curcas. During the past 4-5 years there is an upsurge of activity in promotion of J. curcas in India but castor also holds promise in augmenting and buffering the variation in the tree oilseeds productivity both as sole crop, intercrop or perennial shrub. Castor is naturalized and cultivated in all temperate countries of the world. Castor has the advantage of the availability of world class technologies and varieties and hybrids suited for different climatic conditions. With regard to castor, the world acreage, production and productivity are 1.264 m ha, 1.140 mt and 902 kg/ha, respectively (FAOSTAT, 2006). India with an area of 0.75 m ha, production of 0.73 mt and productivity of 973 kg/ha is the leading producer of castor in the world. Castor is an industrially important crop and has found application in more than 700 products and is being used in lubricants, paints and coatings, cosmetics, engineering plastics and synthetic fibers. Castor has the potential to yield 350-650 kg of oil per hectare under rainfed cultivation and 800-1200 kg oil per hectare under irrigated conditions. Castor has the potential to yield up to 4-5 tonnes per hectare to obtain a maximum of 2000 kg/ha of oil. Estimated CO2 absorption level of castor bean plants is 34.6 tonnes per hectare with 2 growing cycles/year. The use of castor and Jatropha oils as biodiesel has proven to have technical and ecological benefits and provides opportunities for agricultural development in marginal and sub-marginal lands.

Genetic improvement, genetic diversity and molecular markers

Despite the fact that both the crops are ideal candidates for cultivation on marginal lands, both are beset with crop specific problems. According to Jongschaap et al. (4), several claims about J. curcas of low nutrient requirements, low water use, low labour inputs, the nonexistence of competition with food production, tolerance to pests and diseases are not supported by authentic scientific findings. This is absolutely true as most of the information has been with material that is available wild locally. In J. curcas, the varieties are selections from local germplasm and hence, systematic breeding research needs to be initiated for development of varieties suitable for different agro-ecological regions. The breeding objectives include increased seed yield, oil content, increased ratio of female to male flowers, reduced toxicity in seed meal, alteration in tree architecture, and resistance to biotic and abiotic stresses. Jatropha has indeterminate growth habit and should be bred to fit mechanized cultivation and harvesting. J. curcas has the distinct advantage for genetic improvement through interspecific gene transfer. The genus Jatropha encompasses 175 species of which J. curcas is the most primitive species of the genus which facilitates crossing with several species of the genus. Interspecific hybridization coupled with in vitro techniques for overcoming hybrid sterility barriers and manipulation of ploidy can facilitate development of germplasm with increased yield, enhanced oil content, altered fatty acid

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PETROTECH 2009 11 - 15 JANUARY 2009, NEW DELHI, INDIA

composition, value addition to the cake and by-product utilization, improved resistance to pests and diseases, modification of biofuel properties, improved adaptability to problematic sites and also in alteration of ideotype (5). Castor belongs to a monotypic genus, Ricinus and genetic improvement for various traits is based on the variability available in the cultivar germplasm. Germplasm exhibiting extensive genetic diversity is the key for success of the breeding programmes. Studies on use of molecular markers in assessment of genetic diversity are rather limited in castor and confined to use of AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat) markers. Genotyping of J. curcas germplasm using different types of molecular markers, such as, RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeats), AFLP and SSR indicated low inter and intra-accessional variability in local germplasm and maximum divergence between accessions grown in most of the tropical regions versus the accessions from Central American region. Adequate genetic variability exists in castor germplasm which could be exploited in the breeding programmes. However, in case in J. curcas, germplasm from Central American countries need to be used for widening the genetic base. As of now, 322 nucleotide sequences, 13 expressed sequence tag (EST) sequences and 122 protein sequence databases are available in the NCBI database for Jatropha. Likewise for castor, 467 nucleotide sequences, 59251 EST sequences and 303 protein sequence databases are available. Genome sequencing of castor has been initiated at The Institute for Genomic Research (TIGR) to facilitate gene identification and annotation. The haploid chromosome number of castor is ten and the genome has been assembled and sequenced to 4X coverage using a whole genome shotgun strategy (www.tigr.com). While there is a need for augmenting the genomic database for J. curcas, the resources available for castor are adequate for mining the database for gene sequences for different purposes.

Tissue culture and transgenics

In J. curcas, tissue culture protocols for shoot regeneration from seedling tissues and leaves from mature plants through both organogenic and embryogenic pathways are optimized. The media requirements are simple and medium supplemented with the cytokinins benzyl adenine or thidiazuron in combination with the auxin indole-butyric acid facilitates shoot regeneration although the concentrations vary with the genotype and the explanted tissue. Somatic embryogenesis is promoted on medium supplemented with kinetin and indole-butyric acid. The major concern is mass propagation of elite genotypes for obtaining large quantity of quality planting material. J. curcas plants can be propagated through vegetative cuttings but the plants have lower longevity, low clonal multiplication rates, are easily uprooted with strong wind, less resistant to drought to biotic stresses when compared to plants propagated by seeds. For propagation through tissue culture, a reproducible protocol of bud proliferation with acceptable multiplication index, good shoot elongation and rooting is a prerequisite. Reports of shoot proliferation using axillary and apical buds are available but commercial feasibility of these techniques has yet to be demonstrated. Castor proved to be highly recalcitrant to in vitro manipulations and tissue culture regeneration is confined to few tissues and genotypes (6). In J. curcas, genetic transformation through Agrobacterium-mediated method has been reported but development of transgenics harbouring agronomically desirable genes has not been accomplished. In castor, meristem-based shoot proliferation has been used to develop transgenics through both direct and vector-mediated methods. These methods have been tried to develop insect resistant transgenics and ricin-free transgenics (6). Gressel (7) in his review has provided an exhaustive list of genes that could be deployed in to J. curcas for developing genotypes with dwarf plant type, suppressed branching, anti-shattering, reduced levels of curcin and suppressed phorbol ester production. Likewise, in castor, transgenic approach through RNAi technology has been proposed for reduction of the toxic protein ricin and conversion of ricinoleic acid rich castor oil to oleic rich oil.

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PETROTECH 2009 11 - 15 JANUARY 2009, NEW DELHI, INDIA Use as biodiesel

J. curcas oil is rich in oleic acid and has the advantage of being used as straight vegetable oil and also as transesterified oil without any modifications to the engines (Table 1). J. curcas oil has chemical composition and physical properties similar to that of rapeseed oil and is suited for conversion to biodiesel that conforms to the EN14214 standard (8). Castor oil is unique in that 90% of its fatty acid is ricinoleic acid which is a 12-hydroxyoleic acid (Table 1).

Table 1: Fatty acid composition (%) of seed oils of J. curcas and castor
Fatty acid Myristic acid Palmitic acid Stearic acid Arachidic acid Behemic acid Palmitoleic acid Oleic acid Linoleic acid Ricinoleic acid Dihydroxystearic acid Linolenic acid Eicosanoic acid J. curcas oil 0-0.1 9-22 5-8 0-2 0-2 0-1 35-51 27-42 ----Castor oil -1 1 ---3 4.2 89.5 0.7 0.3 0.3

The presence of hydroxyl group and double bonds imparts unique chemical and physical properties that make castor oil a vital industrial raw material and stabilizes the oil against oxidation (9). The fuel specific properties of both the oils are presented in Table 2. Castor oil is the only oil soluble in alcohol and does not require the consequent energy requirement in transesterification as for other vegetable oils. Castor oil has a good shelf life when compared to other vegetable oil and it does not turn rancid when subjected to excessive heat. Castor oil is four times more stable than olive oil. The high viscosity, high water content, higher compressibility than other vegetable oils, reduction of about 10% of hydroxyl and acid values if stored for about 90 days and the premium price of castor oil are the major issues limiting the use of straight castor oil as a fuel for internal combustion engines (10). However, the limit values of viscosity, density and cetane number can be met through transesterification followed by dilution or blending with conventional diesel fuel and other vegetable oils. The key areas of research include development of oleic acid rich castor or production of ricinoleic acid in heterologous systems. It is relatively easy to modify the biosynthetic pathway rather than producing the industrial fatty acids in heterologous systems owing to low level of accumulation of these products in transgenic plants. One such example is the transgenic expression of the fatty acid hydroxylase (Fah12) gene under the control of strong seed specific promoter which resulted in very low levels (<1% in tobacco, 17% in Arabidopsis) of accumulation of ricinoleic acid thus, limiting commercial exploitation (11). Metabolic pathways including those involved in oil accumulation in seeds are conserved across plant species and such information has been utilized to develop designer oil crops. Information on mechanisms in seed-oil accumulation and metabolic engineering of fatty acid profile can be applied for improvement of oil content in J. curcas and oil quality in castor. The expression profiles of genes involved in fatty acid biosynthesis, transport during cellular endosperm development and accumulation of triacylglycerols were investigated in castor (12). Oil content can be increased by introducing/overexpressing the genes. Conversion of ricinoleic rich castor oil to oleic rich castor oil can be accomplished through silencing the fatty acid hydroxylase gene. Glycerine is the coproduct which is in great demand as raw material for cosmetics, medicine and food product industries. Before the economic evaluation of all the various plant parts, commercial development of J. curcas remains a major challenge.

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PETROTECH 2009 11 - 15 JANUARY 2009, NEW DELHI, INDIA Table 2: Fuel specific properties of J. curcas and castor oil
Property Seed oil content (5) Molecular weight Melting point Solidification point Viscosity (dPa.s) Ash content Sulphur Potassium Calorific value (MJ/kg) Iodine value Cetane value (Flammability) Density (g/ml) Flash point Viscosity at 400C (CSt) Acid value Saponification value Kinematic viscosity (400C) mm2s-1 Water content (%) Oxidation stability (lus) J. curcas oil 33-36% ---4.4 <0.01 --38-40 94-102 58.5 0.88-0.92 172-2680C 34.36 1.24-3.8 177-198 --8.39 Castor oil 48-54% 298 50C -120C to -180C 9.5-10.0 <0.02% 0.04% Negligible 39.5 82-90 42 0.956-0.963 2600C ---240-300 0.15-0.30 --

Toxic components in seed meal

The major concern with regard to the use of these two non-edible oilseed crops is the presence of the toxic proteins in the seed meal which creates health problems among growers, processors and consumers. The toxic substances in J. curcas include a lectin (curcin), phorbol esters, saponins, protease inhibitors and phytates due to which the seeds, press cake and the oil of J. curcas cannot be used for human or animal nutrition (13). Ricin, a two-chained polypeptide and Ricinus communis agglutinin (RCA), a four-chained polypeptide are the two major toxic seeds in castor seeds. Increased expression of ricin, 2S albumin and ricinoleate production start at 26 days after pollination (12). Ricin is extremely toxic and has potential for use in bio-warfare and cancer immunotherapy. Chemical detoxification procedures are available but the energy input needed to treat the deoiled seed meal to destroy the toxic proteins and allergens limit the economic competitiveness of the procedures used. In castor, both conventional and biotechnological approaches are being attempted to develop cultivars with reduced levels of toxin. Traditional breeding methods involving crosses of a high yielding dwarf line Hale with 2 lines with low levels of seed toxins resulted in development of a dwarf plant type that showed 75-70% reduction in ricin and RCA toxins (14). Conventional breeding approaches combined with transgenic approach was expected to produce castor plants that have potential for 99.9% reduction in ricin content in segregating generations (15). However, transgenic strategies through antisense silencing met with limited production as ricin production is controlled by multiple genes. Current focus is on Knocking out the genes using classical mutagenesis and TILLING (Targeting Induced Local Lesions in Genomes) which identifies single nucleotide polymorphisms (SNPs) as compared to known sequences of genes responsible for ricin production (www.arcadiabio.com). In case of J. curcas, a non-toxic variety of J. curcas has been found in the Papantla region of Veracruz State in Mexico, which is suitable for human consumption after roasting. The innocuous nature of this J. curcas variety was established using fish and rats as experimental models. Molecular markers that could distinguish the toxic accessions from the non-toxic genotypes are developed. These non-toxic Mexican genotypes have reduced or nil levels of phorbol esters but still contain half the amount of toxic curcin lectins as that of the toxic genotypes, 25% more trypsin inhibitors and 50% more saponins (13).

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PETROTECH 2009 11 - 15 JANUARY 2009, NEW DELHI, INDIA Conclusions

The population growth, ever increasing use of transport fuels, rising prices of fossil fuels, climate changes and environmental pollution demands use of renewable energy sources for a more sustainable energy solution. Vast scope exists for exploitation of J. curcas and castor as bioenergy crops although there are still some technological challenges to overcome. A combination of conventional breeding methods with biotechnological techniques provides newer routes for designing oils for food and biofuel purpose. Modification of fatty acid profile in terms of position and number of double bonds and introduction of desired functional groups can be undertaken to meet the requirements of the biofuels.

References
1. W. Siemers, Global perspectives on biofuels, In: J. Keith Syers, David Wood and Pongmanee Thongbai (Eds). Proceedings of the International Technical Workshop on Feasibility of Non-Edible Oilseed Crops for Biofuel Production. Organised at Mae Fah Luang University, Chiang Rai, Thailand, May 25-27, 2007, pp. 5-16, 2008. 2. J. Heller, Physic nut Jatropha curcas L. Promoting the conservation and use of underutilized and neglected crops, 1. International Plant Genetic Resources Institute, Rome Italy. p. 66. 1996. 3. G.M. Gubitz, M. Mittelbach and M. Trabi, Exploitation of the tropical oil seed plant Jatropha curcas L, Bioresource Technol, 67, 73-82, 1997. 4. R.E.E. Jongschaap, W.J. Corre, P.S. Bindraban and Branderburg WA, Claims and facts on Jatropha curcas L. Global Jatropha curcas evaluation, breeding and propagation programme, Plant Research International B.V. Wageneingen, The Netherlands, Report 158, 2007. 5. M. Sujatha, Prospects for varietal improvement of Jatropha curcas, In: J. Keith Syers, David Wood and Pongmanee Thongbai (Eds). Proceedings of the International Technical Workshop on Feasibility of Non-Edible Oilseed Crops for Biofuel Production. Organised at Mae Fah Luang University, Chiang Rai, Thailand, May 25-27, 2007, pp. 105-118, 2008. 6. M. Sujatha, T.P. Reddy and M.J. Mahasi, Role of biotechnological interventions in the improvement of castor (Ricinus communis L.) and Jatropha curcas L, Biotechnology Advances 26, 424435, 2008. 7. J. Gressel, Trangenics are imperative for biofuel crops, Plant Science, 174, 246-263, 2008. 8. G. Francis, Jatropha as a biofuel crop: potential and issues, In: J. Keith Syers, David Wood and Pongmanee Thongbai (Eds). Proceedings of the International Technical Workshop on Feasibility of Non-Edible Oilseed Crops for Biofuel Production. Organised at Mae Fah Luang University, Chiang Rai, Thailand, May 25-27, 2007, pp. 38-48, 2008. 9. D.S. Ogunniyi, Castor oil: A vital industrial raw material, Bioresource Technol, 97, 1086-1091, 2006. 10. V. Scholz and J.N. da Silva, Prospects and risks of the use of castor oil as a fuel, Biomass and Bioenergy, 32, 95-100, 2008. 11. P. Brown and C. Somerville, Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acid hydroxylase cDNA from castor bean, Plant Physiol, 113, 933-942, 1997. 12. G.Q. Chen, C. Turner, X. He, T. Nguyen, T.A. McKeon and D. Laudencia-Chingcuanco D. Expression profiles of genes involved in fatty acid and triacylglycerol synthesis in castor bean (Ricinus communis L.), Lipids, 42, 263-274, 2007. 13. H.P.S. Makkar, A.O. Aderibigbe and K. Becker, Comparative evaluation of non-toxic and toxic varieties of Jatropha curcas for chemical composition, digestibility, protein degradability and toxic factors, Food Chem, 62, 207-215, 1998. 14. D.L. Auld, S.D. Pinkerton, E. Boroda, K.A. Lombard, C.K. Murphy, K.E. Kenworthy, W.D. Becker, R.D. Rolfe and Ghetie V. Registration of TTU-LRC castor germplasm with reduced levels of ricin and RCA120, Crop Sci, 43, 746-747, 2003. 15. D.L. Auld, R.D. Rolfe and T.A. McKeon, Development of castor with reduced toxicity. J New Seeds, 3, 61-69, 2001.

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