2021-22 Annual Update / Final Report
Client report summary:
Key:
CONT-47267-CRFRP-AGR C10X1603-CR-6
Project:
Forages with Elevated Photosynthesis and Growth
Contract ID:
C10X1603
Investment process:  CRFRP 2016 Contestable Research Fund - Research Programmes
Organisa�on:
AGR AgResearch Limited
IMS assigned to:
Alison Slade
Repor�ng period:
01/07/2021 to 30/06/2022
Contract total value:  $11,500,000.00
Team:

Annual Update
2021-22 Annual Update
As this is the ﬁnal report, this annual update will cover a brief history of the basis for this contract and the science that
led up to the start. The signiﬁcance of each of the ﬁve key impact areas will be described and then how this research
progressed. Finally, the outputs of the research and their signiﬁcance will be discussed, including seven peer reviewed
scien�ﬁc publica�ons and the major ﬁndings as wel  as areas yet to be published. Importantly this contract has had
strong stakeholder support and the signiﬁcance of this to the contract and to the stakeholders will be summarized.
A Brief Science History.
The genesis for this outstanding science originally came from research to increase the energy density of forages in the
early 2000s. The team were developing a faty acid expression and protec�on system in the model species Arabidopsis
(Winichayakul et al., 2013). They successfully doubled leaf fat levels and a serendipitous discovery opened the door
to a wide range of applica�ons in crops.
During analysis of the transgenic Arabidopsis, it was observed that the plants grew 50% faster than control plants.  The
team  iden�ﬁed  that  these  plants  had  elevated photosynthesis. This was subsequently  demonstrated  in  forage
perennial ryegrass. More recently (from research in this contract), it has been discovered that the main mechanism
for enhanced photosynthesis is a reduc�on in the nega�ve feedback based on the carbohydrate status of the plant
(Beechy-Gradwell et al., 2020). It should be noted that over the last two decades, enormous investment has been
made  interna�onally  toward  improving  this  complex  process in plants. Before  this  discovery,  while incremental
improvements were made, other research eﬀorts were limited by this nega�ve feedback mechanism plants use to
regulate photosynthesis.
The discovery of enhanced photosynthesis led to the recogni�on that this novel technology had applica�ons in mul�ple
crops. Over the last 14 years, the team developed new partnerships, progressed the science in other crops, and
contributed to building novel commercializa�on models. Within a few years, this important technology is expected to
beneﬁt farmers and consumers in several countries (soybean in 2026).
During  presenta�ons  to  Dairy  NZ,  PGG-Wrightsons Seeds, Agriseeds and Grasslanz Technology in 2015 it  was
recognized that a joint MBIE-Industry funded research programme could progress the development of HME ryegrass
to a stage where it became a commercializa�on programme. There were important fundamental ques�ons on the
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plant response to nitrogen, its water use, the mechanism for enhanced photosynthesis and the chal enges of breeding
in PC2 containment to be answered. The need for HME ryegrass ﬁeld trials and animal nutri�on trials to establish the
value proposi�on for New Zealand was recognized. As New Zealand had not adopted gene�cal y modiﬁed crops the
social license and farmer engagement was another important ac�vity.
Initiation of the Research Programme for Contract C10X1603.
The research was divided into ﬁve key areas: Carbon Dioxide Recycling, Nitrogen U�lisa�on; Nitrogen and Water Use
Eﬃciency; Breeding in Containment; Increasing Farmer Awareness and understanding of HME Forages. The industry
co-funding supported some of the ﬁve key areas and in addi�on helped support the USA based ﬁeld trials. The plan
was to progress to an animal nutri�on trial in dairy cows but in 2021/2022 the strategy for this changed due to a new
understanding that the USA climate was not suitable and the emerging opportunity of the Australian market.
Carbon Dioxide Recycling.
High metabolizable energy (HME) ryegrass plants have increased levels of lipids stored in the green �ssues in micro-
organelles (Winichayakul et al., 2013; Roberts et al., 2010, 2011; Beechy-Gradwel  et al., 2020). These organelles are
stable  within  the  leaf  and  remain  during  the  ensiling  process  (Winichayakul et al., 2020). The early Arabidopsis
research published in 2013 (Winichayakul et al., 2013) speculated that  CO2  recycling led to the enhanced
photosynthesis. However, as it has turned out from research in this contract, there is another very novel and important
mechanism and this has been one of the most important discoveries from this research.
The al oca�on of diﬀerent sources of carbon (sugars and fat) in diﬀerent �ssues is altered, leading to reduced nega�ve
feedback of photosynthesis (Beechy-Gradwell et al., 2020; Cooney et al., 2020). This enables the overal  plant energy
to be increased due to greater ﬁxa�on of atmospheric CO2. Increased plant growth rates are also observed, although
the rate of increase is aﬀected by compe��on for light in densely packed sward condi�ons.
Interna�onal  research  on  photosynthesis  over  the  last  30  years  has  focussed  on  step-by-step incremental
improvements of this complex 156 step set of interac�ng biochemical pathways. Progress was made in various steps
but there were two overarching nega�ve feedback mechanisms based on the carbohydrate status of the plant and the
plants carbon:nitrogen balance. These nega�ve feedbacks o�en limited the progress made. The discovery by the
AgResearch team from research in this contract iden�ﬁed that one of the nega�ve feedbacks (based on the plant
carbohydrate status) was overcome, this has been a major step forwards. While there are condi�ons where HME
ryegrass can have signiﬁcantly greater growth rates, under compe��on for light and nutrients this diminishes and the
plant allocates the extra photosynthate into greater energy density. The same is seen in soybean with the same
technology where occasionally the plants have greater yields, but in general the enhanced photosynthesis leads to
increased energy density stored in the seed through increased oil and protein composi�on.
Nitrogen U�lisa�on.
The plant nitrogen status was linked to photosynthesis. The nitrogen cycle is an important pathway linked  to
photosynthesis in that it supplies the reductant needed to drive the cycle. It was important to understand the plant
response to diﬀerent forms of nitrogen. It had been observed that the plant responds diﬀerently to reduced nitrogen
compared to controls. For forages, this links into the farm nitrogen cycle which includes added and recycled nitrogen.
Research in this area is split into grasses and legumes with grasses including ryegrass and rice (as a model species), and
legumes including alfalfa and now soybean. The overall goal of this research has been to understand the nitrogen
requirement of diﬀerent species and their responses to diﬀerent nitrogen forms, nitrate, ammonia and urea. We made
signiﬁcant progress in ryegrass and this was published in 2018 (Beechy-Gradwel  et al., 2018). The key ﬁndings were
that HME ryegrass u�lised all three forms of nitrogen, but the greatest growth responses were to reduced forms of
nitrogen (ammonia and urea).
Research on soybean has focussed on nitrogen levels within ﬁeld grown plants and we have demonstrated that plants
have some increased leaf nitrogen but not throughout the early reproduc�ve stages and this appears to be then
transferred into the seed which may account for the increased seed protein in some lines. It appears the legume
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symbiosis is suﬃcient to obtain compe��ve yields and addi�on of nitrogen (anhydrous ammonium) only beneﬁts if soil
N levels are low.
Nitrogen and Water Use Eﬃciency.
The  aim  of  this  research  area was  to  determine  if  HME trait  expression  in  transgenic  plants  alters  plant  nitrogen
metabolism. This goal is diﬀerent from the research in described above on nitrate u�liza�on as it is more encompassing
and  focuses  on  overal   plant  nitrogen  metabolism. We  are  also  examining  water  use  eﬃciency  and  other  stress
responses such as light and temperature.
We performed control ed environment experiments on HME ryegrass event ODR4501 and looked at its ability to u�lize
nitrate, ammonium, and urea. HME ryegrass shoot dry weight increased across the en�re nitrogen supply range
regardless of nitrogen form, whereas the non-GM control ryegrass shoot dry weight did not signiﬁcantly increase
beyond 7.5 mM nitrogen supply. At 10 mM nitrogen supply, HME ryegrass shoot dry weight was 27-34% greater and
root dry weight was 25-45% greater than in the non-GM control ryegrass. Total plant percent nitrogen and the shoot
to root ra�o was lower for plants supplied with nitrate than with ammonium or urea but did not diﬀer between the
non-GM control and HME ryegrass. This suggested that HME ryegrass has a similar nitrogen u�lisa�on eﬃciency and
biomass par��oning.
Of par�cular interest is the legume species alfalfa and soybean. As these species can form symbioses with the nitrogen
ﬁxing bacterium Rhizobium, they are provided with a source of nitrogen in the form of ammonium. Research on
soybean has focussed on nitrogen levels within ﬁeld grown plants and some of this is ongoing and being conducted by
our partner ZeaKal. This links to the observa�on men�oned previously that plants have some increased leaf nitrogen
throughout the early reproduc�ve stages and this appears to be then transferred into the seed which may account for
the increased seed protein in some lines. It suggests that alfalfa and white clover are similarly likely to beneﬁt.
The overall nitrogen u�lisa�on of HME ryegrass is relevant to on farm models currently being used to help establish
the value in farming systems. As the program moves into the commercialisa�on phase from 2022 and beyond, we are
repea�ng our farm and ﬁnancial models informed from the US based ﬁeld trial data and recent work on methane
mi�ga�on experiments from in vitro methane assays.
Breeding in Containment and the Ryegrass Endophyte.
The novel breeding approach based in containment facili�es the team has developed in col abora�on with their seed
company partners has enabled seed to be developed for 5 years of overseas ﬁeld trials and has set the programme up
for much larger scale plan�ng for animal nutri�on trials. The breeding occurs in crossing cages in PC2 containment and
takes 9-10 months per cycle. Once suﬃcient homozygous familied have been developed and characterised it is possible
to scale up to an open ﬂowering and pol ina�on system.
Two methods of plant transforma�on were used with gene gun plants produced prior to the start of this contract and
Agrobacterium plants produced during the contract.  We found the Gene Gun plants had mul�ple loci of integra�on of
the transgene gene cassete and these became problema�c during the breeding phase as they segregated and we
ended up with par�al HME phenotypes. The Agrobacterium system produced a favourable frequency of single copy,
single locus integra�ons and we were able to recreate many HME ryegrass plants that performed much beter in the
breeding phase.  To ensure we has intact single copy integra�ons of the T-DNA we performed whole genome sequence
analysis (an innova�on that became an op�on in 2018/2019). This enabled us to map the integra�ons to the genome
assemble and iden�fy genic and intragenic inser�ons.  Not all events were easy to map as the genome is not complete
and therefore, we adopted a long-read sequencing approach which is s�l  to be ful y completed for some events.
The ryegrass endophyte that lives intercellularly within the plant leaves and stem provides protec�on from various
insect predators of ryegrass. As the transgenic HME ryegrass material was developed out of the plant transforma�on
process it was endophyte free (to prevent contamina�on in �ssue culture). The AR1 and AR37 endophytes were
introduced in the breeding process and an important ques�on was how this symbio�c fungus would survive in a HME
ryegrass plant. This has been answered in containment and ﬁeld-based experiments and we have found no diﬀerence
in transmission from seed genera�on to seed genera�on, no diﬀerence in fungal biomass and a minor reduc�on of
some of the protec�ve alkaloids, although the levels s�ll remain within the seasonal varia�on range. This will be
submited for publica�on in 2022.
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The proposed animal nutri�on trials require one ha of HME ryegrass and one ha of a null control line.  This needs 10
to 20 kg of seed for each treatment to be produced in PC2+ containment. Addi�onal y, the three to four cycles of
breeding need to be performed in elite industry ryegrass germplasm. We developed a unique system in the new PC2+
containment  glasshouses at the Palmerston North Campus.  Using  a  rapid  homozygous breeding protocol that
minimises in breeding depression while minimising the genera�ons required, we have developed the ﬁrst batch of
seed and by February 2023 we wil  have produced the 10-15 kg of seed for use in a trial in Australia.  This enables both
in and oﬀ season seed produc�on. Analysis of seed quality indicated it passes the phytosanitary requirements for
shipping to Australia. Protocols were  co-developed with an industry advisory group and containment measures
supervised by the Ministry of Primary Industries. This is the ﬁrst �me this volume of seed has been produced in
containment from a gene�cal y modiﬁed wind pol enated grass species and highlights the unique facility and capability
that has been developed.
Increasing Farmer Awareness and Understanding of HME Forages.
In  col abora�on  with  NZIER,  AgResearch  conducted  a  survey  on  farmer  decision  making  and  the  key  decision
�meframes for a GM HME ryegrass. Key ﬁndings were:
•  Some farmers are interested in the poten�al beneﬁts of HME ryegrass. They may adopt it within a year or two of
its commercial release. However, they want to know more about how it might perform.
•  They are likely to want informa�on early, through many channels.  The best �me to provide informa�on wil  be
a�er the ﬁeld trials and before product release.  Some farmers want informa�on even earlier.  Given the number
of informa�on sources that farmers use, and the �me frames for decision making, the informa�on should be
available in many formats through many channels.
•  The use of gene�c modiﬁca�on (GM) technology creates addi�onal complexity.  Some farmers will not adopt HME
ryegrass because it is GM.  Other farmers would adopt HME ryegrass but recognise that GM technology is an issue
with consumers and in their supply chains.
•  HME ryegrass would likely be a minority of total pasture.  Among poten�al adopters, HME ryegrass would likely
be one of several cul�vars used.
More Recently a Survey of Gatekeepers and New Zealand Agrifood Exports (Kaye-Blake et al., 2022) was conducted to
understand:
•  Whether there is signiﬁcant gatekeeping behavior in New Zealand export supply chains.
•  Where in the supply chains gatekeeping occurs?
•  Whether GM technology might be expected to trigger gatekeeping behavior.
Interviews and an online survey were conducted with respondents involved in export supply chains for meat and milk
products  from  New  Zealand. The  focus  was  on  percep�ons  of  gatekeeping  behaviour  and  the  impact  of  private
standards on the ability to sell GM food to overseas consumers. Gatekeeping and private standards are methods by
which companies can exert inﬂuence on global value chains. The growth in private standards in par�cular has been
advanced  as  a  limita�on  on  producers  and  their  ability  to  innovate. Although results did suggest that there is
gatekeeping in the export supply chains, they provide litle support for the idea that either gatekeeping or private
standards signiﬁcantly impede New Zealand’s ability to market GM food to overseas consumers. Instead, government
regula�on  and  non-GM demand by  some  consumers  were  more  important  factors. It is planned to publish this
research in the journal New Biotechnology.
New Zealand Based Nutrition Trial.
There is poten�al to perform a sheep nutri�on trial to assess the methane mi�ga�on eﬀects of HME ryegrass from
ensiled HME and Control lines grown in PC2 containment. We focussed on methane reduc�ons as published nutri�onal
studies of ruminants suggest a 5% reduc�on in methane emissions for every 1% increase in dietary fat. We aim to have
experimental resolu�on to measure a 10% reduc�on in methane emissions.
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Previous research on the stability of the faty acids stored in the leaf micro-organelles (MSc Thesis Beechy-Gradwell,
2015) and more recently in the publica�on by Winichayakul et al., (2020) looking at in vitro methane assays using
ensiled ryegrass, suggested ensiling was a viable storage method.
We developed a novel system to grow and ensile ryegrass in batches and store the material for up to 18 months un�l
we have approximately 1000 kg of dry mater needed for a trial. AgResearch funded the project that wil  be conducted
over the next 18-24 months.  This system enables us to generate enough dry mater for both HME and control ryegrass
to conduct the trial in sheep at the AgResearch Palmerston North Campus. This means the ﬁrst animal nutri�on study
of this technology wil  be complete by the end of 2024.
USA Field Trials.
AgResearch was able to partner with the University of Missouri in a ﬁve year collabora�ve program to conduct ﬁeld
trial based assessment of HME ryegrass in regulated ﬁeld trials. The robust but user friendly regulatory system in the
USA enabled analysis of gene�cally modiﬁed ryegrass with two restric�ons, no seed planted in the ﬁeld and no plant
reproduc�on. The  ﬁrst  two  years  involved  assessing  the  environmental  condi�ons  and  developing  protocols  for
transplanta�on  of  glasshouse  germinated  seedlings, comparing space plants vs.  sward  growth  condi�ons  and
preven�on  of  reproduc�on. We  ini�al y  assessed  gene  gun  derived  HME  ryegrass  which  turned  out  to  have
problema�c mul�-loci integra�ons of the HME gene cassete that segregated during the breeding. Therefore in 2019-
2021 we used the superior Agrobacterium derived HME ryegrass. The recent publica�on from the team (Beechy-
Gradwell et al., 2022)  shows  the  primary  beneﬁts  for  HME  ryegrass  (elevated  leaf  fats  and  gross  energy)  reliably
translate from the laboratory to the ﬁeld, as demonstrated across two ﬁeld seasons and under realis�c sward-like
growing condi�ons.
Across  the  2019  and  2020  ﬁeld  seasons,  HME  ryegrass  displayed a 25–34%  increase  in  FA  delivering  up  to
0.5 MJ kg-1 DW higher gross energy and no penalty to biomass produc�on. If successfully converted to ME, this energy
gain  is  250%  greater  than  the  total  ME  gain  achieved  over  the  last  4 decades by  tradi�onal  gene�c  selec�on.
Consequently, these increases are predicted to deliver 30 kg MS cow-1 season-1 (based on FARMAX model ing) and 10%
less methane for the New Zealand farmer.
The  transla�on  of  the  secondary  beneﬁts  for  HME  technology,  elevated  photosynthesis,  and  growth  from  the
laboratory to the ﬁeld were also examined in this paper. The team showed that HME ryegrass could deliver 18% greater
carbon assimila�on and up to 13% higher growth, but only in spaced pots, when light compe��on was low. When
grown in dense swards, in either the laboratory or the ﬁeld, HME ryegrass displayed no increase in photosynthesis or
growth rate. Consequently, this paper provides a comprehensive assessment of the reliability of HME technology’s
primary and secondary beneﬁts, what traits translate to the ﬁeld (increased faty acids and gross energy), and which
do not (increased growth and photosynthesis).
The  ﬁnal  2021  HME  ryegrass  ﬁeld  trial  in  the  USA  analysed  the  performance  of  homozygous  families  and  the
assessment of primary and secondary traits.  Building on the 2019 and 2020 trials that u�lised hemizygous families we
were able to demonstrate that gross energy and plant faty acid composi�on translated from lab to ﬁeld. In this line
we observed a yield penalty over the season however it is unclear if it was a feature of this line or the propaga�on
condi�ons. Our ini�al plan had been to perform animal nutri�on studies in the USA, however the environmental risk
of the hot summers led to a new strategy.
Row Crop Science Innovation.
In addi�on to the breakthroughs in forages, signiﬁcant innova�on in soybeans has led to major beneﬁts.
Transla�ng  from  a  model  species  and  grass  into  a  seed-producing legume required new ways to control  gene
expression. The  �ming  of  gene  expression  has  been  cri�cal,  as  the  goal  is  to  provide  the  plant  with  suﬃcient
photosynthate for eﬃcient seed development. This has been achieved with increased seed oil and protein composi�on
and an overall increase in both co-products per acre over mul�ple seasons and sites across the USA. More recently,
the ZeaKal team have shown that PhotoSeed™ plants can outperform their controls in non-irrigated se�ngs. About
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75%  of  U.S. soybeans are propagated in non-irrigated  condi�ons. A  recent  innova�on  is  the  use  of  genes  and
regulatory elements derived solely from soybean which wil  assist the deregula�on process.
The ZeaKal team has also branched into hemp and developed hemp plants with increased leaf oil. This poten�ally adds
a new applica�on to this crop, one that has lacked the plant breeding developments of other crops due to prohibi�on.
The team has also branched into corn with the poten�al for greater oil yields per acre.
Intel ectual Property.
A cri�cal component for raising investment in biotech is a robust and inves�ble patent por�olio. Since 2009, the team
has developed a por�olio of 13 patents around increasing plant oils, protec�on of plant oils, enhancing photosynthesis,
modifying plant architecture and reducing nitrous oxide emissions from crops. These are granted in mul�ple (92)
jurisdic�ons. Part of the strategy has been extending the life of the patent por�olio by ﬁling more recent methods
patents and new applica�ons. To date, this por�olio has led to the raising of nearly $100M of government, industry,
and venture capital investment. There is poten�al that HME ryegrass may provide farmers with a tool for both methane
and  nitrous  oxide  reduc�ons  and  this  could  be  u�lised  alongside  other  mi�ga�on  measures  to  make  signiﬁcant
reduc�ons to Australasia’s greenhouse gas inventory. The new patent ﬁling extends the protec�on period for this
technology well beyond the 2029 expiry date for the cysteine oleosin patent.
Social and Environmental Beneﬁts.
HME ryegrass represents a unique opportunity to improve both the produc�vity and environmental impact of pastoral
farming. Exempliﬁca�on  of  the  stable  transla�on  in  the  USA  ﬁeld  trials  of  HME  was  a  major  step towards
commercialising this technology and delivering these beneﬁts to farmers both domes�cal y and abroad.
Improving the nutri�onal composi�on of forages through the accumula�on of faty acids in the grazed por�on of the
plant provides signiﬁcant poten�al produc�vity and environmental beneﬁts. A forage with increased metabolizable
energy provides farmers the opportunity to maintain produc�vity using less land and poten�ally fewer animals.  There
also poten�al beneﬁts of reduced methane emissions, supported by evidence from several animal nutri�onal studies
and from in vitro methane assays. The goal is to achieve a minimum of a 10% reduc�on in methane emissions.
The  improved  nutri�ve  quality  is  expected  to  reduce  nitrogen  excre�on  in  ruminants  leading  to  reduced  nitrate
leaching and nitrous oxide emissions. Recently, evidence from control ed environment experiments suggests another
signiﬁcant  reduc�on  in  nitrous  oxide  emissions  can  be  directly  derived  by  the  altered  plant metabolism and
morphology. This can drive up farm proﬁtability and provide farmers with addi�onal tools to manage environmental
impacts from pastoral grazing systems. Experiments in control ed environment chambers on HME ryegrass and control
mesocosms designed to measure nitrous oxide emissions from plants treated with bovine urine have indicated a novel
mi�ga�on poten�al. Two HME ryegrass events were tested in diﬀerent industry cul�vars. In one case a signiﬁcant 50%
reduc�on in nitrous oxide emissions was observed over the course of the experiment in cul�var Impact and in the
second more modern line there was a clear trend for reduc�on. We an�cipate that in most modern cul�vars the
reduc�on would be lower than the 50% seen in CV Impact. This would need to be veriﬁed in ﬁeld condi�ons and may
comprise part of the Australian science plan.
Future Commercialisation Options Beyond the Contract Completion.
We are now developing a commercialisa�on and science plan to progress in Australia in 2023 and are about to seek
regulatory approval from the Oﬃce of the Gene Technology Regulator.  The Australian market is a good opportunity as
there is already GM crop produc�on and there is a signiﬁcant ryegrass market for both the dairy, sheep and beef
industries. Australia can become a market leader to inform New Zealand on the beneﬁts of HME ryegrass in pastoral
agriculture.
Current ac�vi�es include developing the costed dra� science plan, iden�fying the ideal business model from four
possible op�ons, bringing in new partners and fund raising.
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References.
Somrutai Winichayakul, Amy Curran, Roger Moraga, Ruth Cookson, Hong Xue, Tracey Crowther, Marissa Roldan, Greg Bryan, Nick Roberts (2022).
An alterna�ve angiosperm DGAT1 topology and poten�al mo�fs in the N-terminus. Fron�ers in Plant Science, sec�on Plant Proteomics
and Protein Structural Biology. Manuscript ID: 951389 In Press.
Beechey-Gradwel  Z, Kadam S, Bryan G, Cooney L, Nelson K, Richardson K, Cookson R, Winichayakul S, Reid M, Anderson P, Crowther T, Zou X,
Maher D, Xue H, Scot R, Al an A, Stewart A, Roberts N (2022). Lolium perenne engineered for elevated leaf lipids exhibits greater energy
density in ﬁeld canopies under defolia�on. Field Crops Research 275 (108340)
Cooney, L.J., Beechey-Gradwel , Z., Winichayakul, S., Richardson, K.A., Crowther, T., Anderson, P., Scot, R.W., Bryan, G., Roberts, N.J. (2021).
Changes  in  leaf-level  nitrogen  par��oning  and  mesophyl   conductance deliver increased  photosynthesis  for  Lolium  perenne  leaves
engineered to accumulate lipid carbon sinks. Fron�ers in Plant Science 12, ar�cle 641822.
Beechy-Gradwel , Z., Cooney, L., Winichayakul, S., Andrews, M., Hea, S-Y., Crowther, T., and Roberts N., (2020) Storing carbon in leaf sinks enhances
perennial ryegrass carbon capture especial y under high N and elevated CO2. J. Experimental Botany 71:2351-2361.
Winichayakul, S., Beechy-Gradwel , Z., Muetzel, S., Molano, G., Crowther, T., Lewis, S., Xue, H., Burke, J., Bryan, G., and Roberts, N., (2020) In vitro
gas produc�on and rumen fermenta�on proﬁle of fresh and ensiled gene�cal y modiﬁed high-metabolizable energy ryegrass. J. Dairy
Sci 103 (3):2405-2418.
Beechy-Gradwel , Z., Winichayakul, S., Roberts, N., (2018) High lipid perennial ryegrass growth under variable nitrogen, water and carbon dioxide
supply. Proc. NZ Grasslands Assoc. 80:219-224.
Winichayakul,  S.,  Scot,  R.,  Roldan,  M.,  Ha�er,  J-H., Livingston, S., Cookson, R., Curran, A., and Roberts, N. (2013)  In  vivo  packaging  of
triacylglycerols enhances Arabidopsis leaf biomass and energy density. Plant Physiol. 162:626-639.
Roberts NJ, Scot RW, Winichayakul S, Roldan M, (2011). Methods for increasing CO2 assimila�on and oil produc�on in photosynthe�c organisms.
WO2013022053A1, US61/515,610 Roberts NJ, Scot RW, Winichayakul S, Roldan M, (2010). Modiﬁed neutral lipid encapsula�ng proteins
and uses thereof. PCT/NZ2010/000218 WO/2011/053169.

Publicly Available Informa�on.
High Metabolisable Energy (HME) Ryegrass is being developed as an op�on for farmers in New Zealand, and temperate
climates overseas. HME  has  the  poten�al  to  increase  farm  produc�vity  while  reducing  livestock's environmental
impacts, for example nitrogen leaching and methane emissions. Many of the environmental impacts occur because
the propor�on of protein in NZ's forage plants is far in excess of the energy available to livestock. This means that
there is an excess of nitrogen from plant proteins, which is excreted in urine and subsequently lost from the farm
through nitrate leaching. In addi�on, the greenhouse gas methane is produced by methanogenic microbes in the
rumen (stomach) of livestock as they digest the forage.
To improve the nutri�onal balance and increase the overall amount of energy available to livestock on each hectare of
pasture the Team has produced gene�cal y modiﬁed (GM) plants that has specialised microscopic oil micro-organelles
in the leaves of ryegrass. This extra oil, while only being a small propor�on (2 - 3.3%) of the plant's dry mater, delivers
up to a 10% increase in the amount of energy available to an animal ea�ng the plant. This means that an animal can
eat less grass to obtain the energy it needs, then along with that - lower intake means less excess protein/nitrogen in
the urine.
This project was started almost 20 years ago and since then the amount of methane produced by NZ's livestock has
come under na�onal and interna�onal scru�ny due to its impact on increasing global warming. However, along with
reducing nitrogen losses, HME Ryegrass also has the poten�al to reduce methane produc�on. Studies have shown
that livestock diets with higher amounts of oils in their diets produce lower amounts of methane. By matching the
level of oil in our plants against the results from those other studies, it appears that HME Ryegrass has the poten�al to
reduce methane emissions by 10 - 17%. This may not be the 'silver bul et' but when combined with other products in
development it can become part of the bigger solu�on to global warming and climate change.
Serendipitously we also found that under good growing condi�ons HME Ryegrass has enhanced photosynthesis and
growth. All plants have exquisite control mechanisms that al ow them to eﬀec�vely 'snack' on light as needed, where
one of these control mechanisms is the rate of carbohydrate/sugar forma�on going on in the plant. By using the
carbon-based molecules, typical y used to produce carbohydrates, to produce addi�onal oils instead, it appears that
the plant overcompensates, capturing over 20% more carbon dioxide to and conver�ng it into more plant biomass and
energy.
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While it was possible to apply to undertake ﬁeld trials with the GM HME Ryegrass in NZ, it was decided that the
programme would be able to generate the informa�on it required within a shorter �meframe by conduc�ng trials in
the mid-west of the USA. In 2020 we demonstrated that key characteris�cs of heterozygotes (one copy of the HME
Ryegrass transgenes) were similar when measured in PC2 containment growth chambers and glasshouses or the ﬁeld.
In 2021 we demonstrated that homozygotes (two copies of the HME ryegrass transgenes) and again shown that the
even greater increase in oil in homozygous plants (compared to heterozygous plants) translated from lab to ﬁeld for
gross energy and total faty acid content.
Results of the research has been published in seven peer reviewed scien�ﬁc journal ar�cles between 2018 and 2022.
The program is now focussing on commercialisa�on opportuni�es in Australia.
Key Achievements.
1.  Scaled-Up Breeding and Seed Produc�on of GM grasses in PC2+ Containment.
Conduc�ng a meaningful nutri�on study focussed on methane emissions from large ruminants consuming gene�cally
modiﬁed High Metabolizable Energy (HME) ryegrass requires one ha of HME ryegrass and one ha of a nul  control line.
This needs 10 to 20 kg of seed for each treatment to be produced in PC2+ containment.  Addi�onal y, the three to four
cycles of breeding need to be performed in elite industry ryegrass germplasm. We developed a unique system in the
new PC2+ containment glasshouses at the Palmerston North Campus. Using a rapid homozygous breeding protocol
that minimises in breeding depression and the genera�ons required, we are close to achieving 10-15 kg of seed needed
for a trial in Australia.  This enables both in and oﬀ season seed produc�on. Analysis of seed quality indicated it passes
the phytosanitary requirements for shipping to Australia.
Protocols were co-developed with an industry advisory group and containment measures supervised by the Ministry
of Primary Industries. This is the ﬁrst �me this volume of seed has been produced in containment from a gene�cal y
modiﬁed wind pol enated grass species and highlights the unique facility and capability that has been developed.
2.  Comple�on of USA HME Ryegrass Trials and Commercialisa�on Strategy for Australia.
The  ﬁnal  2021  HME  ryegrass  ﬁeld  trial  in  the  USA  analysed  the  performance  of  homozygous  families  and  the
assessment of primary and secondary traits.  Building on the 2019 and 2020 trials that u�lised hemizygous families we
were able to demonstrate that gross energy and plant faty acid composi�on translated from lab to ﬁeld. In this line
we observed a yield penalty over the season however it is unclear if it was a feature of this line or the propaga�on
condi�ons. Our ini�al plan had been to perform animal nutri�on studies in the USA, however the environmental risk
of the hot summers led to a new strategy.
We are now developing a commercialisa�on and science plan to progress in Australia in 2023 and are about to seek
regulatory approval from the Oﬃce of the Gene Technology Regulator.  The Australian market is a good opportunity as
there is already GM crop produc�on and there is a signiﬁcant ryegrass market for both the dairy and sheep & beef
industries. Australia can become a market leader to inform New Zealand on the beneﬁts of HME ryegrass in pastoral
agriculture.
3.  Iden�ﬁca�on and Paten�ng of a Novel Approach to Reduce Agricultural Nitrous Oxide Emissions.
Experiments  in  control ed  environment  chambers  on  HME  ryegrass  and  control mesocosms  designed to measure
nitrous oxide emissions from plants treated with bovine urine have indicated a novel mi�ga�on poten�al. Two HME
ryegrass events were tested in diﬀerent industry cul�vars. In one case a signiﬁcant 50% reduc�on in nitrous oxide
emissions was observed over the course of the experiment and in the second line there was a clear trend for reduc�on.
This would need to be veriﬁed in ﬁeld condi�ons and may comprise part of the Australian science plan. We ﬁled a
provisional patent in 2022 on the use of this technology for reducing nitrous oxide emissions in farming systems. We
have  successful y  obtained  funding  for  a  PhD  studentship  to  further  elaborate  the  mechanisms  for  nitrous  oxide
reduc�ons. There is poten�al that HME ryegrass may provide farmers with a tool for both methane and nitrous oxide
reduc�ons and this could be u�lised alongside other mi�ga�on measures to make signiﬁcant reduc�ons to Australasia’s
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greenhouse gas inventory. The new patent ﬁling extends the protec�on period for this technology wel  beyond the
2029 expiry date for the cysteine oleosin patent.
4.  Development of Forage Ensiling System in PC2 Containment and its Use in Animal Trials.
There is poten�al to perform a sheep nutri�on trial to assess the methane mi�ga�on eﬀects of HME ryegrass from
ensiled HME and Control lines grown in PC2 containment. We focussed on methane reduc�ons as published nutri�onal
studies of ruminants suggest a 5% reduc�on in methane emissions for every 1% increase in dietary fat. We aim to have
experimental resolu�on to measure a 10% reduc�on in methane emissions.
We developed a novel system to grow and ensile ryegrass in batches and store the material for up to 18 months un�l
we have the 1000 kg of dry mater needed for a trial. AgResearch funded the project that wil  be conducted over the
next 18-24 months. This system enables us to generate enough dry mater for both HME and control ryegrass to
conduct the trial in sheep at the AgResearch Palmerston North Campus. This means the ﬁrst animal nutri�on study of
this technology wil  be complete by the end of 2024.
5.  New Knowledge on Plant DGAT1 Enzymes Patented, Published and In Commercialisa�on.
The enzyme diacylglycerol O-acyltransferase 1 (DGAT1) is ubiquitous in all eukaryo�c organisms and has been wel
studied in animals where there is a clear model for its func�on, structure and topology as a membrane bound enzyme.
This contract has supported research inves�ga�ng the func�on of both monocotyledonous and dicotyledonous plant
DGAT1s and the team made a breakthrough understanding the topology of plant DGATs and their func�on. This has
added signiﬁcantly to the understanding of plant DGAT1s which has previously been thought to have diﬀerent topology
to animal DGAT1s. DGAT1 is a fundamental component of the HME technology in ryegrass and it also has value in
increasing oil seed composi�on. The team has recently ﬁled a new provisional patent on a novel mechanism and uses
of plant DGAT1s and published this in Fron�ers in Plant Science. The IP developed in a family of four patents is currently
in commercialisa�on in the USA by AgResearch’s biotechnology start-up partner ZeaKal Inc.  One version may be in the
US market in soybean in 2026 once regulatory approval has been obtained.

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Document Outline