1. BIONIC JATROPHA BREEDING PLATFORM
All pictures of interspecific F1 hybrid courtesy of Ton Rulkens, Flickr
Defining the planting material for Bionic JcL 3.0
© 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA 1

2. ABBREVIATIONS USED
 PoC = proof of concept
 JcL = Jatropha curcas Linn
 Jint = Jatropha integerrima
 Joth = Other Jatropha species
 NTMA = Non-toxic Mexican accession (of JcL)
 PE = Phorbol ester (critical toxin in JcL)
 F1, F2, F3… = generations of hybrids
 BC1, BC2,… = generations of hybrid back crossing with the original
parent population
 FC1, FC2,… = generations of forward crossing with other than parental
accessions/species
 HX(.X) = hybrid family (exact member number)
 HCXXXX = hybrid clone of XXXX
2 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

3. BACKGROUND – GLOBAL RESEARCH
Most important citations from global research literature:
“These data suggest that the chances of improvements in commercially desirable traits of J. curcas are higher through
interspecific breeding rather than the intraspecific breeding approach, which is limited by the low genetic variability in
globally distributed accessions of J. curcas.” (4)
“The studies (…) indicate the limitations of using intra-specific breeding for J. curcas improvement; as exemplified by
the failure to obtain low PE hybrids in crosses between J. curcas toxic accessions from India and the NTMA” (4)
“…analysis of the (JcL x Jint) F1 plants revealed advantages gained through hybridisation (…) including low PE levels.
A back cross with J. curcas NTMA further reduced the PE levels comparable to NTMA – a highly desirable target. Our
analysis revealed higher genetic distance between J. curcas and J. integerrima than between J. curcas accessions.” (4)
“These hybrid (JcL x Jint) clones also expressed superiority in terms of early flowering and fruiting coupled with early
yield, which thus lends scope for further promotion and utilization of Jatropha as a successful biofuel crop.” (6)
“For J. curcas to be a commercially viable source of biodiesel much research and development is needed and little
should be expected from the present widespread planting of wild varieties.” (4)
“If the seed cake were rendered non-toxic and could be used as animal feed, the profitability of cultivating Jatropha,
which was more expensive than diesel in India in 2005, would be „„dramatically increased.‟‟ (5)
3 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

4. BACKGROUND – OWN STUDIES
 Localization is crucial for commercial planting material as the genotype x ecosystem (GxE)
interdependence is high in JcL
 Epigenetics play a critical role for JcL commercialization being the only available sound explanation for
many aspects of the species‟ behavior
 JcL intraspecific hybridization usually results in very high levels of heterosis if parents exhibit
genetic diversity
 Non-toxic, high yielding hybrids are possible and have a series of ground breaking advantages for
the future
 Non-toxic seed cake is a high value animal feed adds an absolute minimum of US$ 500 per ha to
the JcL bottom line
 Reported barriers to hybridization within the Jatropha genus often prove untrue
 Proven breeding paths for JcL are available:
 Heterosis breeding offers quick path to early sucesses
 JcL x Jint has been tried successfully at different instances and shows a high success probability in terms of superior yield
and other favorable traits
 Adding other Jatropha species increases possibilities even further, but very limited successes have been reported
 BPL‟s ongoing proof of concept aims to close knowledge gaps on interspecific breeding routes
 Importance of adding true wild accessions from center of origin has been clarified
 Outlook: There is potential to develop JcL into an annual industrial crop, a route which would resemble
the development of Castor decades ago
4 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

5. BACKGROUND - CONCLUSIONS
 JcL is currently an undomesticated wild plant previously used for living hedges
 Genetic variance within typically (“commercially”) used JcL accessions is too limited (literature)
  The germplasm base needs to be significantly expanded for successful breeding results
 Introduction of high heterozygosity through interspecific crossing Is BPL’s primary route of choice
 J integerima is the species we prioritize, others will follow
 Successful interspecific hybrid paths reported in literature
 Truly wild accessions from Central America offer an alternative, intraspecific breeding approach (heterosis)
 Collected by dedicated expeditions, several have been made available to this program by Geneticlab et. al.
 Genetic analysis shows significantly higher heterozygosis compared to the usual homozygosis found in most “commercial” JcL
 Many are low/non-toxic
 Advantage for time to market due to significant heterosis (hybrid vigor) effects
 Induced polyploidy is an advanced breeding tool we are investigating
 Stable inbred lines of all selected parental material need to be established
 (semi) commercial and wild
 toxic and non-toxic
 selected future hybrid lines
 Advanced genetic technologies need to be applied
 Selection of best suited parental material
 Marker assisted breeding
 Identification of clear gene-trait relationships is the next step
 Breeding for horizontal resistance
 Significantly reduce the cost of pesticides
5 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

6. GENETIC MAPPING SUPPLIES GUIDANCE
Central American landraces
Commercial varieties
(cultivated in Africa and India)
Outgroups
(Jatropha spp.)
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7. APPROACH GREATLY DIFFERS FROM OTHERS
 Nobody can effectively analyze hundreds or even thousands of accessions
phenotypically and genetically (as widely claimed)
 Most accessions are genetically identical or very closely clustered
 In JcL high phenotypic differences don‟t necessarily correlate with high genetic differences
 A wide spread misconception by current JcL breeders
 Reasons not clear yet, but epigenetic effects are the most assumed explanation available
 Effective selectiveness guarantees execution of successful breeding paths
 A small core collection of proven genetically divers material has been established at BPL
 Collection will be expanded by new hybrids and additional wild accessions over time
 Guidance from literature and scientific reports is integrated
 Active investigations for sought after properties will be initiated when required
 A global network with like minded JcL growers/breeders is maintained
 Cooperation with scientific researchers
 Genetic marker analysis and development
 Biochemical analysis
 Integration of latest scientific research findings
 Program accompanied by long term scientific research
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8. PROGRAM OBJECTIVES
 Stringent commercial approach to scientifically based JcL hybrid breeding
 A clear proof-of-concept on the most promising approaches by mid 2012
 Reduced risk
 Reliable planning base
 A fast track success by mid 2013 (first generation non-toxic hybrids ready for field trials)
 Marketable success stories by mid 2012 to support critical funding initiative
 Funding initiative based on proof-of-concept results
 Promising crosses in the early generations have to exhibit genetic improvements for important traits
 Only reproducible improvements backed by genetic analysis are relevant
 Public news flow to be established before the end of the PoC
 Mandatory traits
 Non-toxic
 Further heat treatment for anti nutritional factors will be always required (similar to soya)
 High yielding material for West African ecological environments
 Further preferential traits
 Horizontal pest resistance (mealy bug!)
 Oil content & composition
 Biomass production
 Uniformity in size and shape (mechanical harvesting)
 Synchronous flowering and fruiting cycles (mechanical harvesting)
8 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

9. INTERSPECIFIC JCL HYBRIDS REPORTED
 J curcas x J integerrima
 Many detailed reports
 Verified by BPL
 J gossypifolia x J curcas
 Reciprocal is J tanjorensis (sterile)
 Patented by Nandan
 Verified by BPL
 J gossypiifolia is extremely diverse with many taxonomically close
relatives
 J curcas x J scaposa
 Under testing (Mozambique)
 J curcas x J macrocarpa
 Under testing (Argentina)
9 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

10. Strictly confidential
SYSTEMATIC BREEDING PATH
Variations for XXX
Female parent Pollen donor Intraspecific:
• Genetically remote JcL accessions
JcL • Non-toxic JcL accessions
XXX
starter • Wild accessions from center of origin
Interspecific:
• J integerrima
• Other Jatropha species
F1 Hybrid • hybrids
• ….
BC1
F2 Inbred hybrid lines F2, F3, F4….
BC2 Fx Genetic variance has to be taken
into account via permanent
selection supported by genetic
markers
BC3
10 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

11. Strictly confidential
UPDATE 1: ACTUAL F1 BREEDING PATH
Tested F1 are GHLT x Jint and GHOLD x Jint
Female parent Pollen donor Variations for XXX
Currently performed: future
F1 as female
parent XXX • J integerrima • GHLT
• Other Jatropha • MXCLO
• MXZA • ….
• MXHU
F2/FCF1/BCF1 • Self cross
Hybrid
current future
F1 as pollen • MXCLO8.5/7.10 • Self cross
XXX donor • MXZA • GHLT
• MXHU • More NTMA
• GUA1A1 • ….
F2/FCF1/BCF1 • Other Jatropha
Hybrid • J integerrima
11 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

12. POSITIVE AND PROVEN
Jint JcL Joth
Parental genotypes
H4=JothxJcL
H1=JcLxJint H4=JothxJoth 1st hybrid generations
H0=JcLxJcL interspecific intraspecific
H6=JothxJint
intraspecific
Building up large hybrid populations to enable full exploitation of genetic variability
through selection and cross breeding
H1BC1 (GAL, GHLT, CLO7.10) 2nd hybrid generations
H1F2 (H1.2xH1.2) H1FC1 (H2, GUA, CLO, HU, ZA, ....)
12 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

13. Strictly confidential
UPDATE 2: ACTUAL JOTH RESULTS
Current results
 Joth (green) x Joth (red)  intraspecific
 Joth x JcL (H2)  interspecific
 Jgos x GHLT (H2.1)
 Jgos x MXHU (H2.2)
 Joth x Jint  interspecific
Observations
 Very strong maternal effect
 Hybrids usually difficult to recognize
 Seed texture is a reliable phenotypic indicator in most cases
 Intraspecific hybrids usually express some hybrid vigor (heterosis)
 Leave size, plant height
 Seed size, fruit size
13 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

14. Strictly confidential
PARENTAL MATERIAL FOR CORE APPROACH
 JcL Starter:
 Selection from best Asian, Indian, African, South American semi commercial cultivars
 Selection from non-toxic and wild Central American accessions
 In-crossed accessions:
 Selected non-toxic
 Selected wild (natural heterozygosity)
 Jatropha integerima (heterozygosity)
 Presumed natural hybrid from Cuba (Dehgan)
 Proven good results from a chain of well documented Indian breeding experiments
 Improved growth
>>> We successfully produced 45 different JcLxJint hybrids during the PoC
 Jatropha gossypifolia (heterozygosity)
 Three distinctively different wild accessions identified in Ghana
 Reported success of a fertile JcL hybrid (US patent application)
 Enormous vegetative growth rate (banned as invasive in NSW, Australia)
 Very strong pest resistance
 Favorably shaped for mechanical harvesting
14 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

15. Strictly confidential
VARIATIONS OF CORE APPROACH
 Alternative Starters:
 Jatropha other (reported success)
 Jatropha integerrima
 Non-toxic JcL
 Wild JcL
 Incrossed accessions:
 Selected JcL (non-toxic, wild, commercial)
 Jatropha integerima (heterozygosity)
 Jatropha other (heterozygosity)
 Induced polyploidy
 First line experiments ongoing with several natural polyploid JcL plants available
 Targets:
 New hybrids as parental material with favorable traits
 Non-toxic hybrids with high heterozygosity (primarily as further breeding base)
 Inter- and intraspecific
 Jint/Joth hybrids as new established breeding base (patent applications pending)
15 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

16. TRANSGENIC APPROACH
 Interspecific breeding is a necessity
 Limited genetic variance with globally accessible material
 J integerrima has ideal genetic properties
 A natural hybrid from Cuba with high heterozygosity (Dehgan and others)
 see variability in leaves
 see variable flower colors
 100% heterozygosis reported - to be verified within the program
 Seeds contain high levels of linoleic acid
 Fast growth and permanent flowering (3 months to first flowering reported)
 J other easy intraspecific crossing but difficult for interspecific hybridization
 Enormously vegetative
 Very high horizontal resistance
 Potential for an annual crop similar to Castor development
 Direct crossing with JcL (Dehgan says “impossible”)
>>> we produced a limited number of healthy seeds and seedlings in 2011 for
all combinations listed above, further PoC trials ongoing in 2012 establishing a
sound base for future mainstream breeding
16 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

17. NON TOXIC
 Why not?
 Toxicity does not increase pest resistance
 Some wild non-toxic accessions exhibit superior expressions of
important traits
 A must for acceptable labor conditions
 Modern crop commercialization requires strict health protection for workers
at all times
 Co-carcinogenic effect of PE becomes evident only after years of exposure
 Any kind of detoxification process cannot eliminate that issue
 Fundamental turn around for weak JcL business models
 Press cake forms a high quality animal feed without the need of chemical
detoxification
 Poultry feed
 Fish feed
 Potentially adds 1000 USD and more to a farms revenue stream per ha
17 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

18. TOOLS BEYOND HYBRIDIZATION
 Artificial/natural polyploidy
 Checking farm for more natural occurrences
 Improve experimental procedures
 Use of growth regulators
 GA3 – coconut water
 BA - Benzyladenine
 Number of flowers
 female/male ratio of flowers
 PBZ
 Induce early flowering/fruiting
 Reduction of vegetative growth
 Synchronous plantation cycle
 Other plant enzymes
 Vitamin B1- Thiamin
 In vitro propagation possible for important hybrids
18 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

19. TOOLS BEYOND HYBRIDIZATION (2)
 Dedicated crop cycle management
 Water management
 Abundant for vegetative phase (drip irrigation from reservoirs)
 Mild water stress for flowering/fruiting
 Synchronized pruning
 PBZ as additional control instrument
 Soil amendments & nutrient applications
 Mycorrhiza and other micro organisms show very positive results if applied correctly
 Biochar additions show strong effect on vegetative growth
 Enzyme treatment for faster propagation are under investigation
 Manipulation of day light
 Research required
 Extend to 16 hours for breeding nursery
 Find plausible background first
 Verify assumption: Best areas today are 15 - 20 degrees away from the equator
 Try domestication for 0-15 degrees
 Replication of Mexican daylight times
19 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

20. Strictly confidential
THE BIONIC JCL BREEDING TRIANGLE
oil quality > high domestication > limited
seeds > medium-large genetic variability > low
soil requirement > low uniformity > low
drought resistance > high yield > insufficient
pest resistance > moderate flowering > asynchronous
non-toxic accessions > yes toxicity > medium - low
market acceptance > high
+ _
oil quality > very high Jatropha oil quality > medium
seeds > small curcas Linn seeds > very small
soil requirement > medium soil requirement > low
drought resistance > medium drought resistance > high
pest resistance > low pest resistance > very high
non-toxic accessions > no
market acceptance > none
+ + non-toxic accessions > no
market acceptance > low
Jatropha Other
domestication > none
genetic variability > high
_ integerrima Jatropha _ domestication > none
genetic variability > high
uniformity > no uniformity > high
yield > high yield > very high
flowering > permanent flowering > permanent
toxicity > high toxicity > very high
20 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

21. Strictly confidential
TRAITS TARGETED FOR OPTIMIZATION
 Crop yield
 Seed weight
 Number of flowers & female/male ratio
 Branching
 Natural or pruning induced
 “Willingness to flower” of new branches
 Oil content & quality
 Crop cycles per year
 Quality of crop
 Toxicity
 Oil composition
 Agronomic suitability
 Uniformity
 Canopy shape and size
 Synchronous flowering
 Drought resistance
 Pest resistance
 Horizontal resistance
21 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

22. AN ELITE WILD ACCESSION FROM THE BIONIC COLLECTION
A member of a family of wild germplasm collected from the center of origin
 Non-toxic
 Male sterile
 High yielding based on several distinct traits
• 12-20 fruits per cluster
• 100-seed weight 85+ gram
• Strong, permanent flowering
• Strong branching
 Cuttings develop flowers before leaves
 Similarly new branch shoots from pruning
often start flowers before leaves
22 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

23. OPEN INFORMATION EXCHANGE POLICY
 Bionic is promoting an open exchange policy in Jatropha plant development
 Looking for collaborative partnerships
 Mutually beneficial relationships
 Balanced give and take
 Open source approach where ever possible
 Protected intellectual property only where necessary and useful
 We wish to talk with anyone in the industry
 Open dialog without predjudice
 Explore common industry
 Alignment of objectives
Talk to us....
 exchange@bionic-palm.com
23 © 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA