Research

Pongamia Pinnata Research

 

oilBio Energy Plantations Australia Pty Ltd funded Pongamia research that was conducted by Professor Peter Gresshoff and his team at The University of Queensland. The following article presents the progress of this research and key commercial benefits as at November 2010.

 

Research Background

Our philosophy:

-It is essential for optimal production, and response to changing market or environmental situations to understand the genetic make-up of an organism

15_months_seedpods-Only through in-depth knowledge can genotype-environment interactions be deciphered for sustainable biofuel and biomaterial production

-Biofuel crops should be stress tolerant and nitrogen fixing to avoid competition for agriculturally valuable land/water/labour/fertiliser needed for food and feed

-A tree crop requires less inputs, is more stress-tolerant as it avoids the fragile seedling stage for most of its production cycle

-A biofuel feedstock organism should be non-toxic, non-weedy and adaptable to a wide range of global environmental regions.

 

Our approach:

-BioEnergy Solutions Pty Ltd, in partnership with BioEnergy Plantations Pty Ltd, UniQuest Pty Limited   (the highly successful commercialisation arm of The University of Queensland), and the ARC Centre of Excellence for Integrative Legume Research (www.cilr.uq.edu.au) are developing critical technology platforms for the genetic, genomic and biotechnology application to the legume tree Pongamia pinnata.

 

-The UQ research has attracted large numbers of Honours and postgraduate students, local, state and Federal government funding, and fosters public communication in the field of sustainable bioenergy and biofuel production.

 

-We believe in evidence-based-decision-making, trying to put research data where others place assumptions and long term predictions. This approach is essential to avoid commercial failures as experienced for the introduction of other ‘new crops’.
flowers

C) Pongamia blossoms in November (Brisbane, QLD).

Note that each flower (pea-like) is in a cluster. Several will

abort and fail to make a pod. Each pod contains one

large seed (see “C”) weighing about 1.5 – 2.0 gm when fully

dry. Small green pods are visible within 14 days of flowering.

 

General Properties – Pongamia pinnata

 

•a next generation biodiesel feedstock
• tropical/ sub-tropical growth range
• survives temperatures of -6°C when dormant
• a nitrogen fixing legume; lowered Carbon-footprint)
• high annual seed yield (10-20,000 seed per tree)
• high (35-43% oil content in seed)
• high energy co-products (pod casing, seed starch, protein)
• drought tolerance after sapling stage
• salinity tolerance to 20 dS/m (equivalent to saltbush)
• genetic and genomic knowledge base
• molecular marker technology
• precocious flowering possible in 15 months from seed
• major cropping possible in 4-6 years
• elite germplasm has been identified; clonally propagated
• Further reading: Scott et al (2008) Bioenergy Research 1: 2-11.

nodules

Nodulated root system of a Pongamia plant (4 months old)

Enlargement of a Pongamia nodule section.

Diameter about 3 mm. Note the reddish central

Zone, the site of symbiotic nitrogen fixation.

 

Commercial Benefit of the research:
a) Biofuel production needs to be sustainable with minimal input costs for fertiliser. Nitrogenous fertiliser is the most expensive, and legume plants possess the ability to capture nitrogen from the air and use it for growth and production of seed.

b) It is essential to understand the biology of an organism forming the basis of a new industry. BES through its UQ in-house and other research has developed an extensive database to respond to new challenges.

 

BES-UQ Pongamia Capabilities

•Molecular Biology and Genetics
• Plant Establishment – Silviculture
• Plant Physiology
• Plant Growth Analysis
• Nodulation Analysis
• Nitrogen Fixation Measurements
• Soil-Water Analysis
• Public Outreach – Education
• Research Management
• Industry Interaction
• International and National Networking
• Biotechnology and Genomics

Field Studies and Life Cycle Analysis

 

closely_spaced_plantation 15m_plantation 

above: Closely Spaced Plantation (image on right at 15 months)

28_mplantation

28 month plantation

 

trial_roma

Roma Trial Plantation

 

trials1biomass

Achievements:

• established plantations in several Queensland sites (coastal & inland)
• demonstrated precocious flowering after 15 months (with seed set)
• calibrated 2 year CARBON sequestration, BIOMASS gain and NITROGEN gain
• demonstrated salinity tolerance at 20 dS/m
• tested pruning and planting density regimes
• demonstrated frost survival for growth and flower induction
• demonstrated mechanical harvesting
• collaborated successfully with Coal Seam Gas Industry

Commercial benefits of research:

a)Plantation management and practices needed to be devised and adjusted for Australian and other specific conditions (tropical, nondormant rain-fed [TNDRF], subtropical, winter-dormant rain-fed [STWDRF], subtropical, winter-dormant, irrigated [STWDI].

b) BES-UQ research demonstrated precocious flowering in Pongamia after 15 months from seed. The major benefit is the opportunity to develop a genetics breeding program, which coupled with ‘molecular markers’ and marker-assisted breeding strategies (see later section), allows the development of elite hybrid varieties, sharing superior traits from different Pongamia individuals.

c) LCA (Life Cycle Analysis) is an on-going activity and monitors the inputs and outputs in Pongamia plantation development from seed to full production. BES-research at UQ has monitored Pongamia tree growth (shoots and roots) for 24 months, demonstrating rapid growth and biomass accumulation.

d) LCA and growth analysis show the nutrient requirements associated with superior growth and cropping.

 

Tissue Culture and Clonal Propagation

immature_cotyledonsClonal propagation from immature cotyledons

Commercial benefits of the research:

 

a)Pongamia, based on its outbreeding nature, is highly variable (similar to the human population). To achieve industrial scale production, plants need to be predicable in their growth and cropping behaviour.

b) Elite trees (often called ‘PLUS-trees’ on the Indian subcontinent) can be selected visually by performance and appearance.

c) Clonal propagation can maintain the genetic make-up of an elite tree. Note: this does not imply that the superior character of the tree descendants is maintained as the genotype is likely to interact with any specific environment.

d) Clonal propagation can be achieved by various methods including tissue culture regeneration of vegetative parts of elite trees, rooted cuttings of twigs/branches and grafted branches onto diverse root stock.

e) Different clonal propagation schemes have different genetic and cost advantages.

f) BES-UQ research demonstrated all cloning procedures, though not at a commercial scale. Additional research is required to develop scale-up.

g) Tissue culture propagation lays the basis for gene transfer (GMO).

 

Multiple_bud_culturebud

Multiple Bud Culture from dormant bud Aseptic culture; sterilisation

grafted_saplingrooted_cutting

Grafted Sapling (left), rooted cutting (right) 

 

DNA Marker Technology

•DNA markers have multiple forms and application.
• BES and UQ developed a large range of DNA markers.

 

Applications include:

• diversity testing to assess candidate breeding material
• risk evaluation for ‘Biosafety Australia’ concerns
• plant protection via Plant Variety Rights (PVR) and related IP
• gene isolation via genomics (Map-based cloning)
• association tagging for quantitative trait loci (QTLs)

 

Dna

 

Improved_approach

Marker

Commercial benefits of research:

 

a)BES-UQ research developed a deep Pongamia DNA sequence database of over 2 billion basepairs using SOLEXA deep sequencing. This serves as a knowledge reservoir and library for present and future gene discovery.
b)We have designed DNA markers that permit the analysis of genetic relativeness among individual Pongamia trees. Such knowledge allows one to determine which elite tree to cross (hybridise) with which other tree.

c)   Pongamia DNA marker technology (patent pending) allows the detection of DNA regions linked to regions of economic advantage. This in turn permits molecular breeding.

DNA fingerprinting using DNA markers also facilitates product protection. Elite (or plus-) or similar IP or brand-protected trees can be fingerprinted and protected by plant variety rights (PVR; ‘breeders rights’) protection, avoiding theft of superior genetic material.

 

DNA Deep Sequencing and Gene Discovery

 

Pongamia genomic DNA was deep-sequenced with SOLEXA GAII

 

Millions of ‘read-pairs’ were aligned and annotated using the known

soybean genome sequence (www.phytozome.net/soybean)

 

Regions of similarity were amplified and sequenced from Pongamia.

 

Highlights include:

• sequencing and annotation/alignment of the Pongamia chloroplast DNA (152 kb)
• sequencing and annotation/alignment of the Pongamia mitochondrial DNA (411 kb)
• isolation of Pongamia seed storage protein genes
• isolation of Pongamia oil biosynthesis genes
• isolation of Pongamia oil stability and storage genes
• isolation of photosynthesis and CO2 capture genes
• isolation of all Pongamia mitochondrial energy and growth adaptation genes
• isolation of Pongamia nodulation and nitrogen fixation genes
deep_sequencing

Commercial benefits of the research:

 

“Knowing the chloroplast DNA gives us control of the ‘well-head’ for carbon capture in Pongamia”

 

a)We have a large data base on Pongamia nuclear DNA. Slide 17 shows the results of gene discovery from that dataset. Discovery of genes allows the precise characterisation of heritable traits, critical for Pongamia improvement.

b)   Slide 18 demonstrates the power of deep DNA sequencing. Using the modern SOLEXA technology, we have been able to characterise sufficient Pongamia DNA to assemble, define and sort the coherent length of the circular Pongamia chloroplast DNA (about 152,000 base pairs). The chloroplast is the site of PHOTOSYNTHESIS, i.e., the process by which carbon dioxide (CO2) is captured by the plant, and converted into sugars. Such sugars form the basis for oil and triglyceride biosynthesis.

c) Using the same technology , BES-UQ research has now determined the structure and DNA sequence of the mitochondrial DNA of Pongamia. Mitochondria are the cellular components that harbour the machinery for chemical energy production (RESPIRATION) in all higher organisms. Energy within a cell is critical for growth and adaptation to environmental stress.

d) Genetic improvement is critical for future crop adaptation to diverse geographic, climatic and managements regimes.

 

Gene_expression

 

Commercial benefits of research:

 

a)BES-UQ research defined stages of Pongamia seed development.
b)Critical genes involved in oil (triglycerides) and storage protein synthesis were expression-profiled to determine metabolic bottlenecks.
c)Such bottlenecks will become the target of future metabolic engineering using gene transfer technology. Such technology, involving Agrobacterium-mediated gene transfer and tissue-regeneration, is being developed by BES-UQ under an ARC Linkage project.

 

 

Gene_expression2

Flowering & Seed Properties

 

Pongamia flowers from the second month after the spring equinox.

Some trees flower until the fourth month after the spring equinox.

Pongamia seeds require 8-10 months to mature.

Pongamia seeds may weigh from 1.2 to 2.4 gm (dry weight).

The seed contains from 35-43% oil (as triglycerides)

Up to 55% of the oil is oleic acid (C18:1).

Palmitic (C16:0) and stearic (C18:0) acids are minor constituents (6-9%)

The oil is inedible because of the presence of pongamol and karanjinol.

The storage protein PpSSP50 is a major seed component, related to soybean 7b conglycinin

Further Reading: Scott et al (2008) BioEnergy Research 1: 2-11.

Kazakoff et al (2010) BioEnergy Crops. Royal Society of Chemistry (in press)

Flowering

Commercial benefits of research:

a)The seed is the primary storage organ for biofuel and biomaterial production.
b)Understanding the temporal and spatial distribution of key chemicals and their

biosynthesis machinery is critical for process optimisation.

 

Economic Estimatespongamia

 

•Yield levels per hectare will vary on the environment and management
• Optimal yields of seed per ha per annum are in the 12-15 ton range
• This equates to 5-6 tons of oil per ha per annum (optimal)
• Intermediate yields of seed range from 6-9 tons/ha/annum
• Low yields would range from 0.5 – 4 tons/ha/annum

 

 

• Co-products include: seed cake for soil improvement; pod wall for co-firing or charcoal; leaf/branch for animal feed; honey; health products (oils, astringents); insecticides
• Pongamia in tropical, rain-fed conditions may be harvested by year 4/5
• Pongamia is subtropical rain-fed or irrigated in-land after 4-6 years.
•Pongamia flowering and seed set occurred in Brisbane area by 15 months from seed.
NOTE: Pongamia and Millettia are the same. The latter term was recently introduced

by taxonomists. We prefer to use the established name for the present time.

Millettia is NOT a special Australian, high producing isolate of Pongamia!

 

What Do We Do?
We are a company dedicated to the creation of an economically and environmentally viable alternative to fossil fuels by the use of Pongamia Pinnata (AKA Milletia PInnata).


What Is Pongamia?
Pongamia is a legume tree which is native to Australia and most of the tropical world. We are discovering the many extremes it can handle, including frost, and salty ground, amongst other things.

More importantly pongamia can produce up to 10,000L of oil per hectare, which can be run straight in a slightly modified diesel engine, or converted to biodiesel.


How Can You Grow Pongamia?
Within Australia, we can supply you with seedlings or larger trees, and teach you how to grow them and become a part of this revolutionary industry.

For more information follow the links in the menus.