Jatropha Hybrid Breeding Platform by Bionic Palm, Ghana

© 2011, 2012 Bionic Fuel Knowledge Partners Inc., Oswego, NY, USA

BIONIC JATROPHA BREEDING PLATFORM

Defining the planting material for Bionic JcL 3.0

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All pictures of interspecific F1 hybrid courtesy of Ton Rulkens, Flickr


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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


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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)


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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


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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


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GENETIC MAPPING SUPPLIES GUIDANCE

Commercial varieties (cultivated in Africa and India)

Central American landraces

Outgroups (Jatropha spp.)


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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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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)


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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)


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SYSTEMATIC BREEDING PATH

F1 Hybrid

XXXJcL

starter

Fx

F2

BC1

BC2

BC3

Inbred hybrid lines F2, F3, F4….

Genetic variance has to be taken into account via permanent selection supported by genetic markers

Variations for XXXIntraspecific:• Genetically remote JcL

accessions• Non-toxic JcL accessions• Wild accessions from center of

originInterspecific:• J integerrima• Other Jatropha species• hybrids• ….

Female parent Pollen donor

Strictly confidential


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UPDATE 1: ACTUAL F1 BREEDING PATH

F2/FCF1/BCF1

Hybrid

XXXF1 as

female parent

Variations for XXXPollen donor


Female parent

Tested F1 are GHLT x Jint and GHOLD x Jint

Currently performed:• J integerrima• Other Jatropha• MXZA• MXHU• Self cross

future• GHLT• MXCLO• ….

F2/FCF1/BCF1

Hybrid

F1 as pollen donor

XXXcurrent• MXCLO8.5/7.10• MXZA• MXHU• GUA1A1• Other Jatropha• J integerrima

future• Self cross• GHLT• More NTMA• ….


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POSITIVE AND PROVENParental genotypesJcLJint Joth

1st hybrid generations

2nd hybrid generations

H1=JcLxJintinterspecific

H4=JothxJoth

intraspecific

H1BC1 (GAL, GHLT, CLO7.10)

H1F2 (H1.2xH1.2) H1FC1 (H2, GUA, CLO, HU, ZA, ....)

Building up large hybrid populations to enable full exploitation of genetic variabilitythrough selection and cross breeding

H0=JcLxJcLintraspecific

H4=JothxJcL

H6=JothxJint


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UPDATE 2: ACTUAL JOTH RESULTSStrictly confidential

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 interspecificObservations 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


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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



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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)



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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


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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


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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


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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


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THE BIONIC JCL BREEDING TRIANGLE

Jatrophacurcas Linn

Jatrophaintegerrima

Other Jatropha

oil quality > highseeds > medium-largesoil requirement > lowdrought resistance > highpest resistance > moderatenon-toxic accessions > yesmarket acceptance > high

domestication > limitedgenetic variability > low uniformity > lowyield > insufficientflowering > asynchronous toxicity > medium - low

oil quality > very highseeds > smallsoil requirement > mediumdrought resistance > mediumpest resistance > lownon-toxic accessions > nomarket acceptance > none

oil quality > mediumseeds > very smallsoil requirement > lowdrought resistance > highpest resistance > very highnon-toxic accessions > nomarket acceptance > low

domestication > nonegenetic variability > highuniformity > noyield > highflowering > permanent toxicity > high

domestication > nonegenetic variability > high uniformity > highyield > very highflowering > permanenttoxicity > very high

+ _

+ +

__



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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



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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


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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.... [email protected]

Jatropha Hybrid Breeding Platform by Bionic Palm, Ghana

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