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Developing GLABRA 2 knock out Jatropha curcas plants for increasing seed oil content

Project Proposal

Submitted to

Department of Biotechnology Ministry of Science and Technology, New Delhi

Vittal Mallya Scientific Research Foundation

# 94/3 & 94/5, 23rd Cross, 29th Main, BTM II Stage, Bengaluru– 560 076 Email: vmsrf@vmsrf.org; Web site: www.vmsrf.org

March 2011

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PROFORMA – I PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH AND

DEVELOPMENT, PROGRAMME SUPPORT PART I: GENERAL INFORMATION 1. Name of the Institute/University/Organisation submitting the Project Proposal: Vittal Mallya Scientific Research Foundation 2. State: Karnataka 3. Status of the Institute: DSIR recognized nonprofit organization (Please see Annexure-I) 4. Name and designation of the Executive Authority of the Institute/University forwarding the application: Dr Anil Kush Research Director 5. Project Title: “Developing GLABRA 2 knock out Jatropha curcas plants for increasing seed oil content” 6. Category of the Project (Please tick): R&D (√) / Programme Support 7. Specific Area (Please see Annexure - II): RNA interference

8. Duration: Three Years

9. Total Cost (Rs.) Rs 76, 77,600

10. Is the project Single Institutional or Multiple-Institutional (S/M): S

11. If the project is multi-institutional, please furnish the following: NA

12. Scope of application indicating anticipated product and processes The seed oil content in Jatropha curcas ranges from 28-39%. Increasing the seed oil content in this shrubby tree can alter the economics of biodiesel production. Developing GLABRA 2 knock out plants is expected to increase the oil content by approximately 8 % as seen in Arabidopsis and Brassica. This work will lead to generation of a high oil content Jatropha curcas plants.

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13. Project Summary The seed oil content in Jatropha curcas ranges from 28-40%. Increasing the seed oil content in this shrubby tree can alter the economics of biodiesel production. There are detailed reports in the understanding of biosynthesis of fatty acids and storage lipids, isolation and expression of a number of fatty acid biosynthesis pathway genes for increasing oil content. The role of a master regulator transcription factors like WRINKLED1 (WRI1), GLABRA2 (GL2), LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), PICKLE (PKL) and 6b interacting protein 1-like 1 (ASIL1), has also been shown to effect the oil accumulation in seed. Manipulation of such regulators is believed to be a more effective strategy for increasing oil content than the manipulation of single biochemical steps in plant seed oil biosynthesis. Studies on the GLABRA2 mutant in Arabidopsis show an 8% increase in oil content without effecting the vegetative growth, seed mass and seed number. GLABRA 2 encodes a homeobox protein that is involved in trichome formation, root hair differentiation and seed mucilage production. We propose to develop an RNAi construct of GLABRA 2 driven by seed specific promoter (early embryogenesis) for generating high oil content Jatropha plants. We have been working on Jatropha curcas for past three years, developed a protocol for embryo transformation and developed transgenes for expressing a heterologous phosphatidyl acyltransferase. The plants confirmed by Southern are being maintained under green house conditions for flowering followed by functional assessment. In this investigation we will attempt to increase the oil content in the wild type Jatropha curcas.

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PART II: PARTICULARS OF INVESTIGATORS

Principal Investigator: 14. Name: Dr Puja Ravikumar

Date of Birth: 19.04.1975 Sex (M/F): F Designation: Project Leader

Department: Biological Sciences Institute/University: Vittal Mallya Scientific Research Foundation

Address : 94/3 & 94/5, 23rd cross, 29th main, BTM II Stage, Bangalore

PIN: 560076

Telephone: 080- 26687216 Fax: 080-26687170 E-mail:puja@vmsrf.org Number of research projects being handled at present: One

Co-Investigator 15. Name: Dr. Anil Kush

Date of Birth : September 13, 1956 Sex (M/F) : M

Designation : Research Director

Department : Biological Sciences Institute/University: Vittal Mallya Scientific Research Foundation

Address : 94/3 & 94/5, 23rd cross, 29th main, BTM II Stage, Bangalore PIN : 560076

Telephone : 080- 26687216 Fax: 080-26687170 E-mail : kushanil@vmsrf.org Number of Research projects being handled at present: Two

Co-Investigator 16. Name : Dr. Arti Rani

Date of Birth : February 26, 1978 Sex(M/F) : F Designation : Scientist Department : Biological Sciences

Institute/University : Vittal Mallya Scientific Research Foundation Address : 94/3 & 94/5, 23rd cross, 29th main, BTM II Stage, Bangalore

PIN : 560076 Telephone : 080- 26687216 Fax . 080-26687170 E-mail : arti@vmsrf.org

Number of Research projects being handled at present : One

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PART III: TECHNICAL DETAILS OF PROJECT 16. Introduction

16.1 Origin of the proposal Jatropha seed oil is being used as biodiesel in modern automotive engines. The total seed oil content in Jatropha seeds ranges from 28-40%. Extensive screening in Indian germplasm has shown few high oil yielding varieties with oil content as high as 44%. Current research attempts on improving Jatropha involve increasing the oil content and developing drought tolerant plants. With improvements in the post extraction processing of the seed oil the utility in modern automotive engines will continue to improve. The growing demands for the biodiesel can be met by cultivating high oil content Jatropha. There are reports of increase in oil content in Arabidopsis and Brassica seeds in GLABRA 2 knock out plants. We propose to use the similar mechanism in Jatropha for increasing the seed oil content. 16.2 (a) Rationale of the study supported by cited literature (b) Hypothesis (c) Key questions. Rationale The scientific literature suggests that the seed oil content can be regulated by modifying key enzymes such as ACCase and DGAT in oilseed rape lipid biosynthesis (Roesler et al.1997; Weselake et al. 2007). However, more recent studies have indicated that seed oil accumulation appears to be controlled by a coordinated regulatory mechanism, which is not only pertinent to major steps of lipid metabolism pathways, but also requires coordination of key components in carbohydrate metabolism, in particular the regulation of sucrose and hexose flux (Mu et al. 2008). Therefore, identification of the key genes involved in seed oil content at the level of carbohydrate metabolism represents another strategy for increasing seed oil bioassembly. Screens of Arabidopsis mutants have identified a series of important TFs, which can significantly affect seed oil content. Among these TFs, LEC1 and WRI1 act as master regulators because they appear to regulate the action of other TFs (Weselake et al.2009). The LEC1 gene encodes the CCAAT box-binding domain protein and controls seed oil biosynthesis by inducing expression of more than half of the plastidial fatty acid synthesis genes in Arabidopsis (Mu et al. 2008). The WRI1 gene encodes an AP2/EREB (ethylene responsive element binding) domain protein and promotes oil accumulation by controlling the carbon flux and sugar metabolism in Arabidopsis (Cernac and Benning 2004). In addition to these two TFs, Arabidopsis GL2 (GLABRA 2) gene has been confirmed to play a key role in seed oil biosynthesis (Shen et al. 2006). Chai et al (2010) isolated four distinct orthologues of GL2 from B. napus (AC-genome), B. rapa (A) and B. oleracea (C), using an overlapping-PCR strategy. Suppression of GL2 led to a 2.5 – 6.0 % increase in oil content. Hypothesis Developing GLABRA 2 suppression mutants using RNAi will lead to an incarese in the seed oil content in Jatropha. The suppression will be directed only to seeds to avoid any phenotypic/ vegetative defects.

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Key Questions • Can seed specific promoter driven silencing of GLABRA2 lead to an increase in seed oil

content? • Whether the knock out plants will come display some vegetative / reproductive penalties?

16.3 Current status of research and development in the subject Oil is a major seed storage compound that has significant economic value for food, feed and industrial markets. In seeds, oil is accumulated as triacylglycerols (TAGs), which are synthesized from glycerol-3-phosphate and fatty acyl-CoA in the endoplasmic reticulum. Fatty acids are synthesized from acetyl-CoA exclusively in the plastid, and then transported to the cytoplasm in the form of fatty acyl-CoA (Ohlrogge and Browse, 1995). In the endoplasmic reticulum, TAGs are synthesized by the stepwise acylation of glycerol- 3-phosphate, known as Kennedy pathway. First, fatty acyl moieties are added to the sn-1 and sn-2 positions of glycerol-3-phosphate by glycerol- 3-phosphate acyltransferase and lyso-phosphatidic acid acyltransferase, respectively, to form phosphatidic acid. Phosphatidic acid is then hydrolyzed by phosphatidate phosphahydrase to yield diacylglycerol (DAG). DAG can be used to form TAGs, or it can be used as a substrate for membrane lipid biosynthesis. Diacylglycerol acyltransferase, the only enzyme specific to TAG synthesis, adds a third acyl chain to DAG and yields TAGs (Voelker and Kinney, 2001). Finally, TAGs are stored in seeds in specialized structures termed oil bodies. Each oil body contains a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with structural oleosin proteins (Huang, 1992). Despite a detailed understanding of the biosynthesis of fatty acids and storage lipids, and the isolation of many genes in the pathways, the control mechanism for fatty acid biosynthesis and lipid accumulation in seeds is still not well understood. One in vitro fatty acid feeding study indicated that the supply of fatty acids is a limiting factor for oil accumulation in developing embryos (Bao and Ohlrogge, 1999). Acetyl CoA carboxylase (ACCase), the first enzyme in fatty acid synthesis, is considered to be a control point for carbon flux into fatty acid synthesis (Ohlrogge and Jaworski, 1997). Expression of Arabidopsis cytosolic ACCase in B. napus resulted in a 5% increase in seed oil content (Roesler et al., 1997). However, the individual overexpression of other key enzymes in the fatty acid biosynthetic pathway in plants, such as 3-ketoacyl synthase (KAS I, KAS II, and KAS III), did not lead to an increase in seed oil content (Ohlrogge and Jaworski, 1997). Additional studies indicated that TAG synthesis could be a limiting factor for oil accumulation. In one study, expression of a yeast sn-2 acyltransferase in Brassicaceae increased seed oil content by 20% (Zou et al., 1997). In addition, overexpression of diacylglycerol acyltransferase in Arabidopsis resulted in an 11–28% increase in seed oil content (Jako et al., 2001). More recently, metabolic flux control analysis indicated that there is no single major step controlling oil biosynthesis (Ramli et al., 2002). Approximately 60% of flux control is exerted by fatty acid synthesis, while 40% of flux control was associated with TAG synthesis. These authors suggested that manipulation of a single step in the overall pathway would have little effect on storage lipid accumulation in seed. Identification and modification of regulatory genes that control oil accumulation in seed could be a more effective strategy to increase seed oil content. Plant breeding studies have demonstrated that

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seed oil content can be increased significantly by selection. For example, following 28 cycles of selection, maize kernel oil increased to 20% in high oil ASK (Alexho Single Kernel) lines compared to the initial population with only 4% kernel oil content (Alexander, 1988). Identifying genetic factors that regulate oil accumulation in seed is a key step toward understanding and manipulating storage lipid accumulation in this organ. Several transcription factors involved in seed development may also have pleiotropic effects on seed storage product accumulation. LEAFY COTYLEDON 1 (LEC1) encodes an HAP3 subunit of the CCAAT binding factor and plays a key role in Arabidopsis embryo development (Meinke et al., 1994; Lotan et al., 1998). Deletion mutation in LEC1 resulted in a reduction of seed oil content. Ectopic expression of LEC1 in vegetative tissue initiated formation of embryo-like structures and induced the expression of embryo-specific RNAs encoding oleosin. PICKLE (PKL) is another regulatory gene that controls the expression of LEC1 in vegetative tissues (Ogas et al., 1999; Dean Rider et al., 2003). In a pkl mutant, primary roots accumulated seed storage proteins and oil. WRINKLED1 (WRI1) is the third transcription factor involved in the regulation of seed oil accumulation (Focks and Benning, 1998; Cernac and Benning, 2004). In Arabidopsis, a mutation in WRI1 caused a reduction in seed oil accumulation, while ectopic expression of WRI1 led to increased oil content. Shen et al (2006) isolated a high oil mutant, p777from an activation tagged population in Arabidopsis. They showed that the high-oil phenotype in p777 is caused by the disruption of the GLABRA 2 (GL2) gene, which is a known homeobox protein required for leaf trichome and root hair development. Transcription factors regulate gene expression by directly binding the cis-acting regulatory elements of target genes via their DNA-binding domains or by interacting with other transcription factors. GLABRA1 (GL1), an R2R3 MYB transcription factor, GLABRA3 (GL3), a basic helix–loop–helix (bHLH) transcription factor, and TRANSPARENT TESTA GLABRA1 (TTG1), a WD40 protein, are believed to form a transcriptional activator complex to control the transcription of GLABRA2 (GL2), which in turn induces trichome formation in shoots. Wang et al (2008) reported that disruption or deletion of the DNA-binding domains in either GL1 or GL3 completely abolishes the transcriptional activity of the GL1–GL3 complex in activating GL2.

References Alexander, D.E. (1988) Breeding special nutritional and industrial types. In: G.F. Sprague and J.W. Dudley (Eds.), Corn and Corn Improvement, American Society of Agronomy, pp. 869–880.

Bao. X, Ohlrogge J. (1999) Supply of fatty acid is one limiting factor in the accumulation of triacylglycerol in developing embryos. Plant Physiol. 120(4): 1057–1062. Cernac A, Benning C (2004) WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis. Plant J 40:575–585 Chai C, Bai Z, Wei F, King GJ, Wang C, Shi L, Dong C, Chen H, Liu S. (2010) Brassica GLABRA2 genes: analysis of function related to seed oil content and development of functional markers. Theor Appl Genet 120:1597–1610

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Dean Rider, S. Jr., Henderson, J.T., Jerome, R.E., Edenberg, H.J., Romero-Severson, J. and Ogas, J ( 2003) Coordinate repression of regulators of embryonic identity by PICKLE during germination in Arabidopsis. Plant J. 35(1): 33–43. Focks, N. and Benning, C. (1998) wrinkled1: A novel, low-seed oil mutant of Arabidopsis with a deficiency in the seed specific regulation of carbohydrate metabolism. Plant Physiol. 118(1): 91–101.

Huang AH. (1992) Oil bodies and oleosins in seeds. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 177–200.

Jako C, Kumar A, Wei Y, Zou J, Barton DL, Giblin EM, Covello PS, Taylor DC. (2001) Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol. 126(2): 861–874. Lotan, T., Ohto, M., Yee, K.M., West, M.A., Lo, R., Kwong, R.W., Yamagishi, K., Fischer, R.L., Goldberg, R.B. and Harada, J.J. (1998) Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells.Cell 93(7): 1195–1205.

Mu JY, Tan HL, Zheng Q, Fu FY, Liang Y, Zhang JA, Yang XH, Wang T, Chong K, Wang XJ, Zuo JR (2008) LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in Arabidopsis.Plant Physiol 148:1042–1054 Meinke, D.W., Franzmann, L.H., Nickle, T.C. and Yeung,E.C. (1994) Leafy Cotyledon Mutants of Arabidopsis. Plant Cell 6(8): 1049–1064. Ohlrogge J, Browse J. (1995) Lipid biosynthesis. Plant Cell 7: 957–970.

Ohlrogge J., Jaworski JG. (1997) Regulation of fatty acid synthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:109–136

Ogas, J., Kaufmann, S., Henderson, J. and Somerville, C. (1999) PICKLE is a CHD3 chromatin-remodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis. Proc. Natl. Acad. Sci. USA 96(24): 13839–13844 Roesler, K, Shintani, D, Savage, L, Boddupalli, S. and Ohlrogge, J (1997) Targeting of the Arabidopsis homomeric acetyl-Coenzyme A carboxylase to plastids of rapeseeds.Plant Physiol. 113: 75–81.

Ramli, U.S., Baker, D.S., Quant, P.A. and Harwood, J.L. (2002) Control analysis of lipid biosynthesis in tissue cultures from oil crops shows that flux control is shared between fatty acid synthesis and lipid assembly. Biochem. J. 364: 393–401. Roesler K, Shintani D, Savage L, Boddupalli S, Ohlrogge J. (1997) Targeting of the Arabidopsis homomeric acetyl-Coenzyme A carboxylase to plastids of rapeseeds. Plant Physiol. 113: 75–81.

Shen, B, Kerstin W. Sinkevicius, David A. Selinger A, Mitchel, Tarczynski C ( 2006) The homeobox gene GLABRA2 affects seed oil content in Arabidopsis. Plant Molecular Biology 60:377–387 Voelker KT (2001) Variations in the biosynthesis of seed-storage lipids. Annu. Rev. Plant. Physiol. Plant Mol.Biol. 52: 335–361. Weselake RJ, Taylor DC, Rahaman MH, Shah S, Laroche A, McVetty PB, Harwood JL (2009) Increasing the Xow of carbon into seed oil. Biotechnol Adv 27:866–878

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Wang S, Chen JG. (2008) Arabidopsis Transient Expression Analysis Reveals that Activation of GLABRA2 May Require Concurrent Binding of GLABRA1 and GLABRA3 to the Promoter of GLABRA2. Plant Cell Physiol. 49(12): 1792–1804 Weselake RJ, Shah S, Taylor DC, Zou JT, Laroche A, Moloney MM, Rakow G, Raney JP, Harwood JL (2007) Transformation of Brassica napus with diacylglycerol acyltransferase1 results in increased seed oil content. In: Benning C, Ohlrogge J (eds) Current advances in the biochemistry and cell biology of plant lipids. Aardvark Global, Salt Lake City, pp 232–234 Zou J, Katavic V, Giblin EM, Barton DL, MacKenzie SL, Keller WA, Hu X, Taylor DC. (1997) Modification of seed oil content and acyl composition in the Brassicaceae by expression of a yeast sn-2 acyltransferase gene. Plant Cell 9: 909–923.

16.4 The relevance and expected outcome of the proposed study Transgenic Arabidopsis and Jatropha plants with down regulated expression of GLABRA 2 will be developed. This is expected to lead to an increase in total seed oil content. 16.5 Preliminary work done so far In vitro Regeneration in Jatropha We have developed a protocol for infection of Jatropha embryos followed by in vitro regeneration.

Fig 1: In vitro regeneration in Jatropha using embryo culture We have also done a multiple sequence alignment of the GLABRA2 sequneces from the oil seed plants available in the NCBI database. Degenerate primers for isolating the truncated 550 bp GLABRA2 fragment have been designed and the PCR conditions optimized. The cloning of this fragment is underway.

17. Specific objectives

• Isolation of GLABRA 2 ORF ( truncated and full length) from Jatropha curcas • Developing dsRNAi construct using an intronic linker and driven by seed specific

phaseolin promoter • Assembling the construct in p CAMBIA 1302 • Transforming model plant Arabidopsis for evaluating the effect of dsRNAi construct

by seed oil analysis • Transforming Jatropha curcas embryos • Molecular evaluation of the transgenes

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Fig 2 : Multiple sequence alignment of GLABRA sequences from Brassica rapa, Brassica oleracea and Arabidopsis thaliana

18. Work Plan:

18.1 Work plan Isolation of GLABRA 2 ORF :The GLABRA 2 nucleotide information from NCBI database will be used for designing degenerate primers to isolate the gene from Jatropha curcas. Full length ORF is expected to approximately 2Kb (based on the multiple sequence analysi. Total RNA from the developing embryo tissues of Jatropha will be extracted using traditional phenol RNA extraction protocol followed by column purification. The first strand cDNA will be synthesized by oiligo dT priming followed by PCR using the degenerate primers. The PCR product would be cloned in T vector and sequenced for confirmation.

Developing the dsRNAi construct : A region of approximately 0.7 Kb will be used for developing the dsRNAi construct. The assembly with 0.7 Kb antisense GLABRA 2, a linker of 0.5 Kb and 0.7 Kb sense GLABRA 2 fragment will be made in a cloning vector pBluescript. The linker we plan to use is a 500 bp intron from a terpene synthase from vegetable crop tomato. The assembly will be mobilized in pCAMBIA 1302 that has a seed specific phaseolin promoter ( already available in our laboratory) previously cloned in it.

Mobilizing the recombinant plasmid in Agrobacterium: The recombinant plasmid will be transferred to the Agrobacterium strain LA 4404 via electroporation. The Agrobacterium

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colonies harboring the plasmid will be confirmed by PCR and used for the Jatropha transformation work.

Effect of GLABRA2 dsRNAi in model plant Arabidopsis:We will take up floral dip transformations of the model plant Arabidopsis to assess the effect of the dsRNAi in improving the seed oil content and if it leads to any phenotypic effects. The ovary and developing seeds of the transformed Arabidopsis plants will be studied for any cellular defects by making 10µ microtomal sections. The seed oil content will be estimated using Hexane extraction followed centrifugation at 15,000 rpm for 10 mins. The centrifuged mass will be transferred to hood to evaporate hexane. The extracted sample will be baked for 5 mins at approximately 100°C and the oil content will be estimated by weighing. We will attempt to do the NMR based oil estimation as an outsourcing activity.

Transformation of the Jatropha with GLABRA2 dsRNAi construct:Jatropha embryos will be dissected and sterilized under aseptic conditions. The dissected embryos will be mildly injured using a needle and incubated with Agrobacterium culture for 30 mins with 10µM acetosyringone. The infected embryos will be maintained on acetosyringone medium for 2 days for co cultivation followed by washing with water and Cefataxime (250 µg/ml) and transferred to selection medium for direct shooting. The shoots surviving on the Hygromycin (50 µg/ml) selection medium will be transferred to the rooting medium after three weeks. The plants will be transferred to soil rite and maintained in green house till further analysis.

Confirmation of the Jatropha transgenes: The Jatropha trangenes will be confirmed by PCR and Southern analysis using the leaf tissues. The down regulation of seed specific promoter driven GLABRA 2 will be taken up after three years when the transformed plants flower and set seeds.

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Schematic representation of the work plan 18.2 Connectivity of the participating institutions and investigators: Not Applicable

18.3 Alternate strategies We propose to take up embryo transformations. As an alternate strategy for transformation we will also attempt to take up leaf disc transformations followed by in vitro shooting and rooting.

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19. Timelines:

Period of study Achievable targets 6 Months

Isolation of GLABRA 2 ORF, cloning, sequence conformation , cloning the linker region and developing dsRNAi construct

12 Months Mobilizing the dsRNAi construct in Agrobacterium LA4404, floral dip transformation of Arabidopsis plants, leaf disc and embryo transformation of the Jatropha curcas embryos.

18 Months Regular transformations of Jatropha embryos, tobacco leaf discs and Arabidopsis inflorescence. Maintenance of transformed plants, seed collection from the transformed Arabidopsis plants.

24 Months Inoculation of the Arabidopsis T0 seeds on selection medium, selecting the transformed Arabidopsis plants and maintaining the plants in growth chamber, maintenance of transformed Jatropha plants, Regular transformations of Jatropha tissues. Southern for transgene confirmation in T1 Arabidopsis plants.

30 Months

Seed collection from the transformed Arabidopsis plants, T1 seed inoculation in selection media, New rounds of transformations of Jatropha embryos, hardening of the in vitro rooted plants and transfer to the green house. Seed Oil content estimation in the T2 Arabidopsis seeds

36 Months

Southern confirmation of the transformed Jatropha plants, maintenance of transformed and southern confirmed plants in green house

20. Name and address of 5 experts in the field Sr.No. Name Designation Address 1. Dr S R Bhat Principal

scientist NRCPB, Indian Agricultural Research Institute, Pusa Campus New Delhi 110012.

2. Dr Mohan Singh Principal Scientist

Indian institute of Pulses Research, Kanpur 208024.

3. Dr KK Sharma Principal Scientist

ICRISAT, Hyderabad 500030.

4. Dr. Ramesh Aggarwal Deputy Director

Centre for Cellular & Molecular Biology Uppal Road, Hydrabad– 500 007.

5 Dr. Jitendra Kumar Director Research

G.B Pant University Ranichauri P.O. 249 199, Distt. Tehri, Uttaranchal Pantnagar.

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PART IV: BUDGET PARTICULARS

Budget (In Rupees) A. Non-Recurring (e.g. equipments, accessories, etc.) S. No. Item Year 1 Year 2 Year 3 Total 1 2 3 4

Microtome Tissue water bath Hot air over Plant Growth chamber

700,000 100,000 200,000 20,00,000

Sub-Total (A) = Rs 29, 00,000 Justification: Microtome, tissue water bath and hot air oven will be required for preparing microtome sections of transformed Arabidopsis ovary and developing embryos. Growth chamber will be used for maintaining Arabidopsis plants.

B. Recurring B.1 Manpower (See guidelines at Annexure-III) S. No.

Position No.

Consolidated Emolument

Year 1 Year 2 Year 3 Total

1 SRF (2) Rs. 16000 pm + HRA 30% of the basic

4,99,300

4,99,300 4,99,300

14,97,600

2 Laboratory Assistant (2)

Rs 7,500/ month 1,80,000

1,80,000 1,80,000 5,40,000

Sub-Total (B.1) = Rs 20,37,600 Justification: Two SRFs will be required to take up the transformations and analysis work. B.2 Consumables S. No.

Item

Quantity Year 1 Year 2 Year 3 Total

1 2 3

Fine Chemicals Glasswares Green/ net house house items

As required

400,000 200,000

50,000

400,000 200,000

50,000

400,000 100,000 50,000

11,00,000 500,000 150,000

Sub-Total (B.2) = Rs 17, 50,000 Other items Consolidated

Emolument Year 1 Year 2 Year 3 Total

B.3 Travel 30,000 30,000 30,000 90,000 B.4 Contingency 300,000 300,000 300,000 900,000 B.5 Overhead Sub-total of B (B.1+B.2+B.3+B.4+B.5)

47,77,600

Grand Total (A + B) 76,77,600

Total Budget Rs 76, 77,600

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PART V: EXISTING FACILITIES Available equipment and accessories to be utilized for the project: Few important types of equipment only listed Sl. No. Name of equipment Make Funding agency Year of

Procurement 1 Maxi cold Pharmacia VMSRF 1991 2 Rotavapour Buchii VMSRF/DBT 1995 3 CO2 Incubator Nuair DBT 1998 4 Fluid bed dryer Allience VMSRF 1998 5 Tubular centrifuge CEPA VMSRF 1999 6 Biospectro

Photometer Varian VMSRF 1999

7 Incubator shaker NBS VMSRF 2000 8 Elisa Reader Softmax DBT 2000 9 Spectrophotometer Varian VMSRF 2000 10 Lyophilizer Virtis DBT 2002 11 HPLC Shimadzu VMSRF 2003 12 Laminar flow hoods Alpa VMSRF 2004/2006 13 Gel documentation UVI Tech DBT 2006 14 GC MS Shimadzu VMSRF 2006 15 Milli Q system Millipore DBT 2006 16 Spray Drier Pawan

Engineering VMSRF 2007

17 Micro balance Sartorius VMSRF 2007 18 GC FID Shimadzu VMSRF 2007 19 Light Microscope Olympus VMSRF 2008 20 Preparative HPLC Waters VMSRF 2000

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PART VI: DECLARATION/CERTIFICATION It is certified that

a) the research work proposed in the scheme/project does not in any way duplicate the work already done or being carried out elsewhere on the subject.

b) the same project proposal has not been submitted to any other agency for financial support.

c) the emoluments for the manpower proposed are those admissible to persons of corresponding status employed in the institute/university or as per the Ministry of Science & Technology guidelines (Annexure-III)

d) necessary provision for the scheme/project will be made in the Institute/University/State budget in anticipation of the sanction of the scheme/project.

e) if the project involves the utilisation of genetically engineered organisms, we agree to submit an application through our Institutional Biosafety Committee. We also declare that while conducting experiments, the Biosafety Guidelines of the Department of Biotechnology would be followed in toto.

f) if the project involves field trials/experiments/exchange of specimens, etc. we will ensure that ethical clearances would be taken from concerned ethical Committees/Competent authorities and the same would be conveyed to the Department of Biotechnology before implementing the project.

g) it is agreed that any research outcome or intellectual property right(s) on the invention(s) arising out of the project shall be taken in accordance with the instructions issued with the approval of the Ministry of Finance, Department of Expenditure, as contained in Annexure-V.

h) we agree to accept the terms and conditions as enclosed in Annexure-IV. The same is signed and enclosed.

i) the institute/university agrees that the equipment, other basic facilities and such other administrative facilities as per terms and conditions of the grant will be extended to investigator(s) throughout the duration of the project.

j) the Institute assumes to undertake the financial and other management responsibilities of the project.

Signature of Project Coordinator Signature of Executive Authority (applicable only for multi-institutional projects) of Institute/University with seal Date : Date : Signature of Principal Investigator : Date :

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PRINCIPAL INVESTIGATOR Name : Dr. Puja Ravi Kumar Designation: Research Scientist Department Institute/University Vittal Mallya scientific Research Foundation Date of Birth 19th April 1975 Sex (M/F) F SC/ST : N.A Indicate whether Principal Investigator / Co-investigator: Co investigator Address: Vittal Mallya Scientific Research Foundation, # 94/3& 94/5, 23rd Cross, 29th main, BTM II Stage, Bangalore- 560076. Phone: 91-80-26687216, 26687223 Fax: 91-80- 26687170 Email puja@vmsrf.org Education (Post graduation onwards & Professional career)

Research Experience in various Institutions (If necessary, attach separate sheets) DBT post doctoral fellowship availed 2003-2005 Post Doctoral fellow at Indian Institute of Science, Department of Microbiology and Cell Biology, Bangalore, India with the fellowship sponsored by Department of Biotechnology, Government of India since November 2003 Publications (Numbers only) Books: Nil Research Papers, Reports 5 General Articles Nil Patents: Nil Others : Nil Research Highlights: Dr. Puja has worked on the fatty acid engineering in Indian mustard, a major oilseed crop in northern India. A critical enzyme of the fatty acid synthesis pathway was studied which revealed that engineering thioesterase can lead to production of designer oils. Fatty acid elongase genes were isolated from sub genomic and genomic libraries and the point mutations were detected which lead to LEAR (low erucic acid varieties). Gas Liquid chromatography of the seed oil from exotic germplasm was undertaken to detect any novel fatty acids. In the postdoctoral work, she has worked on flower development in model monocot plant, rice. Ectopic over expression and under expression of homeotic genes was undertaken using an Agrobacterium mediated approach. The targets of the Transcription factors over expressed were detected using semi quantitative RTPCR. She has also undertaken training for Microarray by Affymetrix Inc, USA. List of important publications relevant to the proposed area of work: (Attached) Research Articles Dani, K.G.S, Hatti, K.S, Ravikumar,P and Kush,A ( 2010) Structural and functional analyses of a saturated acyl ACP thioesterase , type B from immature seed tissue of Jatropha curcas. Plant biology doi:10.1111/j.1438-8677.2010.00410.x ( In press)

Sl. No.

Institutional Name Degree Awarded

Year Award/Prize/Certificate

1 Indian Agricultural Research Institute & Devi Ahilya University

Ph. D 2002 SRF by Indian Council of Agriculture Research

2 Devi Ahilya University M.Sc. 1997 II position in University 84.2%

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Raghava T., Ravikumar P., Hegde R. and Kush A (2010) Spatial and temporal secondary metabolite response of tomato cultivars to herbivory and mechanical injury. Plant Science 179(5): 520-526 Dani, K.G.S, Ravikumar,P, Kumar, P.R., and Kush A (2011)Genetic variation within and among small isolated populations of Santalum album - Implications for its conservation. Biologia Plantarum (In press ) Dani, K.G.S, Ravikumar,P, Kumar, P.R., Dani, K.G.S, Ravikumar,P, Kumar, P.R., and Kush A (2010)Genetic Diversity Estimates in Three Geographically Isolated Populations of Santalum album in India. Sandalwood Research Newsletter, 24:1-6 Raghava T, Ravikumar P, Hegde R, Karunakara A.C. and Kush A (2009) Effect of insect herbivory on the volatile profile of tomato cultivars, Karnataka Journal of Agricultural Sciences, 22(5):1023-1028 Rao,N.N, Prasad.,K., Ravikumar.,P. and Vijyaraghavan.,U ( 2008) Distinct regulatory role for RFL, the rice LFY homolog , in determining flowering time and plant architecture. Proc. Natl. Acad. Sci. USA.105(9):3646-3651 Yadav P, Bhat S. R. and Chopra V. L. (2003). Cloning, sequencing and characterization of FAE1 genes of Brassica juncea cv Pusa Bold and the mutant fae1 of B. rapa cv Tobin. J. Plant Biochemistry and Biotechnology, 12: 19-23. Book Chapters Singh,R.K., Khan,S.K., Garg,N., Ravikumar,P. and Chauhan,G.S (2008) Recent advances in Soybean Genomics and Bioinformatics. In: Application of Genomics and Bioinformatics in Plants. Eds Rao,G.P., Wagner,C and Singh,R.K. stadium Press LLC, Texas, USA 389-417 Kush.,A and Ravikumar,P (2007) Oiling the mind to market: customized oils from oilseed crops. In Changing global Oilseed scenario: issues and challenges before India. Indian society of Oilseed Research. Ed Hegde,D.M. pp 65-86 Ravikumar,P., Annadurai,R.,S. and Kush,A ( 2006) Chemical and transcriptomal analysis of herbivore induced volatile signaling in Lycopersicon esculentum. VII Annual Discussion Meeting In Entomology : semiochemicals in crop protection : Ongoing Technologies. Singh.,R.K.,Kumar,P.R., Satyawathi.,T. and Anita .,R.(2004) Biotechnological advances in Soybean .In "Soybean Production and improvement in India". ICAR publication. Eds. Singh,N.B.,Chouhan .,V.,Vyas.,A.K. and Joshi,O.P. pp 47-63. Conference Abstracts Ravikumar P, Manjula P, and Kush A (2009) Chemical and genetic suppression leads to increased shikimic acid accumulation in tobacco BY2 cells. Poster presented in IFS/OPCW workshop “Chemistry in nature – natural resources: Chemical, biological and environmental aspects” organized in Chiang Mai, Thailand from 7th to 10th December 2009. V Karthik, K S Nagesh, A Anantharaj, P Praveen, R Puja (2009) Truncations of PAX9 and Nonsyndromic Oligodontia – An Indian perspective. International Journal of pediatric Dentistry, 19 (1): 32-33 Raghava.T., Ravikumar.P., Hegde,R. and Kush, A ( 2008) In “Advances in metabolic profiling” organized in Lisbon, Portugal ,16-17th 2008.. Differential metabolomic response of Tomato hybrids to insect herbivory. Best Poster Award for poster presentation Ravikumar,P., Annadurai,R.,S. and Kush,A ( 2006) Chemical and transcriptomal analysis of herbivore induced volatile signaling in Lycopersicon esculentum. VII Annual Discussion Meeting In Entomology: semiochemicals in crop protection: Ongoing Technologies.

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Project(s) submitted/being pursued/carried out by Investigator:

Highlights of progress of the project(s) to date (in 200 words) for ongoing projects only (if necessary attach separate sheets) Place: Date: Signature of Principal-Investigator

Sl. No.

Title of Project Funding Agency

Duration From To

No. of Scientists/Associates Working under the project

Total Approved cost of the Project (in Rs.)

1 Development of fermented functional food from papaya

DBT May 2010 to April 2013

two 34.5 lakhs

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CO-INVESTIGATOR Name: Dr. Anil Kush Designation: Research Director Department/Institute/University: Plant Biotechnology Indicate whether Principal Investigator/Co-investigator: Co-Principal Investigator Address: Vittal Mallya Scientific Research Foundation, 94/3&94/5, BTM II stage, Bangalore - 560076 Date of Birth: September 13 1956 Sex (M/F): M SC/ST: N/A

Education (Post graduation onwards & Professional Career)

Sl. No. Institutional Name Degree Awarded Year Award/Prize/Certificate 1 Manipal University MBA 2004 First Division 2 Paris University

(Pasteur Institute) Ph.D 1986 Tres Honorable

Research Experience in various Institutions (if necessary, attach separate sheets)

• Post Doctoral Research Fellow at the Rockefeller University, NY, USA • Post Doctoral Research Associate, Harvard University, MA, USA • Principal investigator, Institute of Molecular and Cell Biology (IMCB), Singapore • Director, (Biotechnology), Indo-American Hybrid seeds, Bangalore • Executive Vice president, Reliance Life Sciences, Mumbai • Director, Vittal Mallya Scientific Research Foundation, Bangalore

Publications Books: 3 (Chapters); Research Papers & Reports: 50 General Articles: 5 Patents: 8 Others: Nil

List of important publications relevant to the proposed area of work: (Attached)

1. Dani, K.G.S, Hatti, K.S, Ravikumar,P and Kush,A ( 2010) Structural and functional analyses of a saturated acyl ACP thioesterase , type B from immature seed tissue of Jatropha curcas. Plant biology ( In press) doi:10.1111/j.1438-8677.2010.00410.x 2. Raghava T., Ravikumar P., Hegde R. and Kush A (2010) Spatial and temporal secondary metabolite response of tomato cultivars to herbivory and mechanical injury. Plant Science 179(5): 520-526

3. Dani, K.G.S, Ravikumar,P, Kumar, P.R., and Kush A (2010)Genetic variation within and among small isolated populations of Santalum album - Implications for its conservation. Biologia Plantarum (In press ) 4. Dani, K.G.S, Ravikumar,P, Kumar, P.R., and Kush A (2010)Genetic Diversity Estimates in Three Geographically Isolated Populations of Santalum album in India. Sandalwood Research Newsletter, 24:1-6 5. Raghava T, Ravikumar P, Hegde R, Karunakara A.C. and Kush A (2009) Effect of insect herbivory on the volatile profile of tomato cultivars, Karnataka Journal of Agricultural Sciences, 22(5):1023-1028 6. Chandregowda V, Kush A, Reddy GC (2009) Synthesis and invitro antitumor activities of novel 4-anilinoquinazoline derivatives. European Journal of Medicinal Chemistry 44,

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3053-3062 7. Chandregowda V, Kush A, Reddy GC (2009) Synthesis of benzamide derivatives of anacardic acid and their cytotoxic activity. European Journal of Medicinal Chemistry 44, 2711-2719 8. Kaushik H S , Chandregowda V, Venkateswara RG, Kush A, Reddy G C (2009) In-silico Interaction Studies of Quinazoline Derivatives for their Inhibitory Action on Both Wild and Mutant EGFRs. J Proteomics Bioinform 2: 126-130. 9. Kaushik H S , Diwakar L, Rao G V , Kush A , Reddy GC (2009) Aromatase Abyssinones and related flavonoids as potential steroidogenesis modulators. Bioinformation 3(9):399-402 10. Pravin Kumar R, Manoj MN, Kush A, Annadurai RS (2007) In silico approach of azadirachtin binding with actins. Insect Biochem. Mol Biol. 2007 Jun;37(6):635-40 11. Prakash AP, Kush A, Lakshmanan P, Kumar PP. (2003) Cytosine methylation occurs in a CDC48 homologue and a MADS-box gene during adventitious shoot induction in Petunia leaf explants.J Exp Bot. 54 (386):1361-71. 12. Singla SL, Pareek A, Kush AK, Grover A. (1998) Distribution patterns of 104 kDa stress-associated protein in rice.Plant Mol Biol;37 (6):911-9. 13. Janicke RU, Porter AG, Kush A. (1998) A novel Arabidopsis thaliana protein protects tumor cells from tumor necrosis factor-induced apoptosis. Biochim Biophys Acta. 12; 1402 (1):70-8. 14. Gidrol X, Sabelli PA, Fern YS, Kush AK.(1996) Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress. Proc Natl Acad Sci U S A.;93 (20):11268-73 15. Sabelli PA, Burgess SR, Kush AK, Young MR, Shewry PR.( 1996) cDNA cloning and characterisation of a maize homologue of the MCM proteins required for the initiation of DNA replication. Mol Gen Genet. ; 252 (1-2):125-36. 16. Adiwilaga K, Kush A. (1996) Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis).Plant Mol Biol., 30 (5):935-46 17. Gidrol X, Lin WS, Degousee N, Yip SF, Kush A. (1994 Accumulation of reactive oxygen species and oxidation of cytokinin in germinating soybean seeds Eur J Biochem. ;224(1):21-8. 18. Gidrol X, Chrestin H, Tan HL, Kush A.( 1994) Hevein, a lectin-like protein from Hevea brasiliensis(rubber tree) is involved in the coagulation of latex. J Biol Chem.;269(12):9278-83. 19. Kush A, Brunelle A, Shevell D, Chua NH.(1993) The cDNA sequence of two MADS box proteins in Petunia. Plant Physiol. 102 (3):1051-2. 20. Chye ML, Tan SA, Tan CT, Kush A, Chua NH (1991) Nucleotide sequence of a cDNA clone encoding the precursor of ribulose-1,5-bisphosphate carboxylase small subunit from Hevea brasiliensis (rubber tree).Plant Mol Biol. 16 (6):1077-8. 21. Chye ML, Kush A, Tan CT, Chua NH. (1991) Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Hevea brasiliensis. Plant Mol Biol. 16 (4):567-77 22. Broekaert I, Lee HI, Kush A, Chua NH, Raikhel N (1990) Wound-induced accumulation of mRNA containing a hevein sequence in laticifers of rubber tree (Hevea brasiliensis).Proc Natl Acad Sci U S A ;87 (19):7633-7. 23. Kush A, Goyvaerts E, Chye ML, Chua NH.(1990) Laticifer-specific gene expression in Hevea brasiliensis (rubber tree).Proc Natl Acad Sci U S A. ;87(5):1787-90.

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24. Kaminski PA, Norel F, Desnoues N, Kush A, Salzano G, Elmerich C.(1988) Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene.Mol Gen Genet. ;214 (3):496-502. Project(s) submitted/being pursued/carried out by Investigator

Sl. No.

Title of Project Funding Agency

Duration From To

No. of Scientists/ Associates working under the Project

Total approved cost of the Project (in Rs.)

1 Genomics and proteomics of Santalum ablum to study the santalol production.

DBT May 2010 to April 2013

1 RA 1 SRF

Rs. 66.25 lakhs

2 Optimizing the seed oil biosynthesis machinery in Jatropha curcas for biofuel production

DBT Aug 2007 to Aug 2011

2 SRF Rs. 36.43 lakhs

Highlights of progress of the project(s) to date (in 200 words) for ongoing projects only Both projects are on metabolomics and genetic engineering of important plant systems. In the Jatropha Project, it is being tried to genetically modify the fatty acid biosynthesis mechanism to produce the seed oil which can be more efficiently used in automobile engines. The proof of concept of the genetic assembly has been developed in Arabidopsis thaliana and transformations on Jatropha are underway. Place: Bangalore Date: Signature

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CO-INVESTIGATOR Name: Dr. Arti Rani Designation: Research Scientist Department/Institute/University: Vittal Mallya Scientific Research Foundation, Bengaluru Date of Birth: 26.02.1978. Sex (M/F): F SC/ST: N/A Indicate whether Principal Investigator / Co-investigator: Co-investigator Address: Vittal Mallya Scientific Research Foundation, # 94/3& 94/5, 23rd Cross, 29th main, BTM II Stage, Bangalore- 560076. Phone: 91-80-26687216, 26687223 Fax: 91-80- 26687170 Email: arti@vmsrf.org Education (Post graduation onwards & Professional Career) Sl. No.

Institutional Name Degree Awarded

Year Award/Prize/Certificate

1 Institute of Himalayan Bioresource Technology, Palampur (H.P.)

Ph.D 2008 CSIR Fellowship

2 H.P.University, Shimla (H.P.)

M.Phil. (Botany)

2001 Gold Medalist

3 H.P.University, Shimla (H.P.)

M.Sc., (Botany)

2000 -

Publications (Numbers only) Books/Chapters---Nil Research Papers: 5, Reports & General Articles ---- Patents: 3 Others: Nil.

List of important publications relevant to the proposed area of work: (Attached)

1. Arti Rani, Kashmir Singh, Payal Sood, Sanjay Kumar and Paramvir Singh Ahuja (2009). P-Coumarate:CoA ligase as a key gene in the yield of catechins in tea [Camellia sinensis (L.) O. Kuntze]. Funct Integr Genomics 9:271–275.

2. Kashmir Singh, Sanjay Kumar, Arti Rani, Ashu Gulati and Paramvir Singh Ahuja (2009). Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea. Funct Integr Genomics 9(1):125-34.

3. Kashmir Singh, Arti Rani, Asosii Paul, Som Dutt, Robin Joshi, Ashu Gulati, Paramvir Singh Ahuja, and Sanjay Kumar (2009). Differential display mediated cloning of anthocyanidin reductase gene from tea (Camellia sinensis) and its relationship with the concentration of epicatechins Tree Physiol 29: 837 - 846.

4. Kashmir Singh, Arti Rani, Sanjay Kumar*, Payal Sood, Monika Mahajan, Sudesh Kumar Yadav, Bikram Singh, Paramvir Singh Ahuja (2008). An early gene of flavonoid pathway, flavanone 3-hydroxylase, exhibits a positive relationship with catechins content in tea (Camellia sinensis (L.) O. Kuntze). Tree Physiology 28(9):1349-1356.

5. Singh K, Raizada J, Bhardwaj P, Ghawana S, Rani A, Singh H, Kaul K and Kumar K (2004). 26s rRNA-based internal control gene primer for reverse transcription polymerase chain reaction-based quantitative expression studies in diverse plant species. Anal

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Biochem 335: 330-333. PATENTS 1. Sanjay Ghawana, Kashmir Singh, Jyoti Raizada, Arti Rani, Pardeep Kumar Bhardwaj &

Sanjay Kumar. Applied 2004, Filed 30.03.06 0344 NF 2004/IN. A method for rapid isolation of RNA & kit thereof.

2. Sanjay Ghawana, Kashmir Singh, Jyoti Raizada, Arti Rani, Pardeep Kumar Bhardwaj and Sanjay Kumar. filed USA 12/295001 dated 29.09.2008. A cost effective less hazardous two solution system for rapid isolation of RNA.

3. Pardeep Kumar Bhardwaj, Arun Kumar, Amit Kishore, Sanjay Ghawana, Arti Rani , Kashmir Singh, Harsharan Singh, Ravi Shankar Singh, Hitesh Kumar, Payal Sood, Som Dutt, Sanjay Kumar, Paramvir Singh Ahuja. A method for cloning functional gene of copper/zinc superoxide dismutases using oligonucleotide primers. (0035nf2008). Filed in India 28th March, 2008.

SEQUENCES OF DIFFERENT GENES SUBMITTED TO NCBI DATABASE 1. DQ194356. Camellia sinensis cultivar UPASI-10 4-coumaroyl CoA ligase mRNA,

complete cds. 2. DQ120521. Camellia sinensis cultivar UPASI-10 chalcone isomerase mRNA, complete

cds. 3. ABA40923. Flavonoid 3',5'-hydroxylase [Camellia sinensis]. 4. EF423762. Camellia sinensis cv. UPASI-10 acetyl CoA carboxylase (ACC)

mRNA, partial cds. 5. AY907710. Camellia sinensis caffeine synthase mRNA, complete cds. 6. ESTs of Arabidopsis obtained through SSH library screening were submitted to GenBank

at NCBI as per the accession numbers ES323544 to 323615. 7. ESTs of tea obtained through SSH library screening were submitted to NCBI GenBank at

NCBI as per the accession numbers ES323490 to ES323543. Place: Bangalore Signature Date