CSIRO Plant Industry

The principal objective of this policy linkage and impact assessment project is to maintain and extend the scientific connections between Chinese and Australian rice research programs, particularly those focusing on tolerance to low temperatures.
Low temperatures during rice crop establishment and reproductive growth cause significant limitations to rice yields and production in Australia and China, and the development of varieties with enhanced levels of cold tolerance is rated the highest priority objective of the NSW DPI rice breeding program.
Additionally, tolerance to low temperatures is a critical foundation for the development of alternative irrigation strategies for rice production, which are aimed at reducing total water use and increasing water productivity.
We conducted an international workshop of Australian and Chinese scientists to review the current status of rice cold tolerance research and to discuss the potential for cooperative projects on cold tolerance. The workshop was to provide the basis for discussion with the objective of initiating joint activities on cold tolerance research.
The workshop was held from December 3rd to 8th, 2006, and the international participants are listed in Table 1. Participants from China were funded by from the project, and scientific critical mass was further enhanced by the addition of three participants from Korea and Japan funded by the Rural Industries Research and Development Corporation
Table 1. International participants to the Rice Cold Tolerance Workshop held at Yanco, December 3-8, 2006

Location
Participant
Address
Comments
Yunnan province, China
Dr Tan Xuelin
Rice Research Institute, Yunnan Agricultural University, Kunming
Rice researcher
Yunnan province, China
Dr Zhao Xiaochun
Rice Research Institute, Yunnan Agricultural University, Kunming
Now located at Sydney University, Cobbity
Heilongjiang province, China
Dr Zhang Fengming
Heilongjiang Academy of Agric. Sciences
Rice Breeder
Beijing
Prof. Wang Huaqi
Upland Rice Research Centre, China Agricultural University, Beijing
Aerobic rice development program
Beijing
Dr Zhikang Li
China Academy of Agricultural Science
Rice Molecular Breeding
Yunnan province, China
Dr Ye Changrong
School of Land and Food Sciences, University of Queensland
From China but now at University of Queensland
Korea, IRRI
Dr K.K. Jena
National Institute of Crop Science, RDA Suweon, Republic of Korea
IRRI representative in Korea
Miyagi Prefecture, Japan
Dr Kuniaki Nagano
Furukawa Agricultural Experiment Station
Rice Breeder
Hokkaido, Japan
Dr Yutaka Sato, Crop Cold Tolerance Research Team
National Agricultural Research Center for Hokkaido Region
Biotechnologist and Team Leader

The foremost outcomes from this meeting are summarised as follows:

Ongoing linkage to rice cold tolerance research in Yunnan province is essential due to the extensive genetic diversity available and the existence of reliable cold tolerance screening facilities at high altitude sites with the area available to test large populations
Development of a relationship with the Upland Rice Research Centre at China Agricultural University is warranted to interact in the development of aerobic rice. While this was not the main focus of the workshop, aerobic rice is important for water-limited areas of China and for future Australian rice production. Aerobic rice requires cold tolerance because there is no protection from standing water. Aerobic rice suffers a small yield penalty in China if adapted cultivars are used. The penalty is likely to be much greater in Australia, however, because humidity is so low. It is unlikely that cultivars could be directly transferred from successful programs in China or Brazil, but knowledge of traits which confer adaptation to aerobic conditions and access to relevant germplasm will be essential.
Germplasm exchange with research programs in the far north of China will be initiated. Production in Heilongjiang Province has increased dramatically from around 100,000 ha in 1949 to 2 million ha in 2006 with a predicted area of 3 million ha within the next decade. This production is entirely japonica rice and varieties are developed using cold-water screening. Unique germplasm characteristics such as the extensive use of semi-erect panicle types also merit investigation.
Cold tolerance is primarily associated with temperate rice production areas and japonica rice is used extensively for its adaptation to cooler regions. At the conclusion of the workshop Dr Jena (IRRI representative, Korea) proposed that a Temperate Rice Research Consortium (TRRC) be developed to continue and improve the liaison between producers of japonica rice. Seed funding for this initiative is to be provided by the Rural Development Administration of South Korea. All participants at the workshop supported the concept, and cold tolerance was seen to be a significant focus of the TRRC.

The principal activity of this project during the period covered by this report was the completion of a study tour to five locations in China. Participants in the study tour included Dr Laurie Lewin, rice industry consultant, Dr Peter Snell, Rice Breeder, Mr Russell Ford, Manager, Rice Research Australia and delegate to the RIRDC Rice Research Committee, and Mr Leigh Vial, rice grower and delegate to the RIRDC Rice Research Committee.
The visit encompassed five locations in China, including Yunnan Agricultural University, Kunming, Guangxi Academy of Agricultural Sciences, Nanning, China Agricultural University, Beijing, Liaoning Academy of Agricultural Sciences, Shenyang and Heilongjiang Academy of Agricultural Sciences, Harbin.
The study tour highlighted the importance of Yunnan as the centre of origin of japonica cold-tolerant rice, and the novel genes for cold tolerance which exist in the genetic background of traditional varieties. Following the visit the strategy for the future is to develop collaborative projects which seek to identify new genes for adaptation to low temperature and their mechanism of action such that new varieties can have multiple sources of cold tolerance.
Much work has been carried out on developing cold-tolerant varieties in a number of improvement programs world-wide, however pedigree analysis reveals that many varieties are built on the same sources of cold tolerance, thus combining varieties may be fruitless if the original gene/s for tolerance are present in each of the parents.
A shift in emphasis to the identification of cold tolerance genes in landraces gathered from high altitude areas in Yunnan is warranted. Although ambitious, new research should be brought to bear on uncovering the genetic basis for cold tolerance in landraces and traditional varieties from cold, high altitude areas, so that these genomic regions can be transferred into other genetic backgrounds to provide robust tolerance at all growth stages.
A total of 740 varieties from the Oryza rufipogon collection in Nanning, Guangxi Province, have been screened for cold tolerance at both the seedling stage and at the reproductive stage, resulting in 4 varieties showing seedling stage tolerance and 2 varieties with reproductive stage tolerance. A backcrossing program has been initiated to transfer tolerance from the wild background into the Australian variety Millin, with regular screening throughout development. This project has not received direct funding and thus is of lower priority with efforts proceeding in the background. Given the location of the institute in the southern part of China where production of indica types predominates, cold tolerance accords lower research priority. Ideally future research should be directed at elucidating the mechanism and genomic location of O.rufipogon cold tolerance genes in comparison with those of Yunnan O.sativa landraces.
There is a need to further explore the origins and mechanisms of cold tolerance in varieties utilised throughout the Liaoning and Heilongjiang provinces and develop the linkages between research institutions. The Heilongjiang Academy of Agricultural Sciences has a cold water screening facility at Harbin in which groundwater at 7C is mixed with surface irrigation water to obtain the desired temperature for screening segregating material.
Finally, further collaborative linkages are warranted with the comprehensive rice breeding program of China Agricultural University aimed at aerobic growing conditions. This research focuses on the development of varieties tolerant of water-limited conditions at all growth stages. Although not specifically aimed at cold tolerance, strategically these are high priority traits to build into future varieties as the Australian Rice Industry faces the prospect of generally lower and less reliable rainfall.
Technology to firstly identify genomic regions identified as contributing to cold tolerance and then to facilitate the efficient transfer of such regions will be increasingly important. Diversity Array Technology (DArT) is one means of obtaining an immediate representation of the genome, and by associating genotype with phenotype, regions associated with cold tolerance can be identified. Sequencing the DArT clones identified with specific positive regions allows identification of the location within the genome and suggests candidate genes for additional study. Further, these areas can be tracked with DArT analysis throughout subsequent crosses and back-crosses made to transfer the traits into varieties with appropriate adaptation and grain quality attributes. Elements of this work are being carried out with segregating populations varying for cold tolerance already developed at Sydney University, however additional funding is necessary to advance the work and enhance the linkages with Yunnan Agricultural University.

This project was originally scheduled for completion in June 2008, however a "no-cost" extension was sought and granted in May 2008.
The primary reason for the extension was that the ongoing drought in southern New South Wales has significantly disrupted normal research activities, and this has limited the achievement of the final project milestones.
The project has three main milestones, which are:
1. To compare the aims and methodologies of rice cold tolerance in Australia and China, by convening a rice cold tolerance workshop with invited scientists from a range of research institutions and locations in China
2. To explore synergies between Chinese and Australian cold tolerance research aimed at production of new varieties with enhanced cold tolerance and grain quality, including technologies such as wide hybridisation, micro-arrays, new genetic technologies and genome profiling methods such as Diversity Arrays.
3. To extend and build new relationships for cold tolerance research, and facilitate exchange of technologies and genetic material.
The first milestone was completed in December 2006 with a successful cold tolerance workshop which involved visits to Sydney University, Cobbitty, CSIRO Plant Industry Canberra, CAMBIA Canberra, and Diversity Arrays in Canberra, Yanco Agricultural Institute, Yanco NSW and Rice Research Australia Pty Ltd, Jerilderie NSW. As a result of this workshop opportunities were explored for linkages between existing complementary research projects in Australia and China, and prospects for new projects and collaboration were discussed. However, nothing was initiated at the time due to extremely limited research funding as a result of a number of years of low production in Australia.
The third and final milestone has two components, the first being a study tour of a range of research institutions in China, undertaken in September 2007, which was the subject of the annual report submitted in May 2008. Briefly the study tour identified a number of strategic initiatives which could be developed further. These included:
extending the links between Yunnan Agricultural University, Sydney University, Cobbitty and Yanco Agricultural Institute for investigation of the genetic basis of cold tolerance in Yunnan rice landraces from high altitude regions
evaluating under NSW conditions, aerobic rices developed at China Agricultural University in Professor Wang Huaqi's program, to determine the key physiological traits which confer adaptation to water limited conditions, and whether these will translate to NSW rice soils
exchange of germplasm with Heilongjiang Academy of Agricultural Sciences in northern China, where they have a dedicated cold water screening system for testing cold tolerance. This would be useful to benchmark our lines against their most cold tolerant varieties
The second component of this milestone was a subsequent visit from Chinese scientists to review Australian research and to finalise progression of these strategic initiatives. The aim was to host a visit from Chinese rice scientists to the Australian rice industry at harvest (March-April 2009) to review our breeding program and selection protocols, as well as on-farm trials and off-site screening nurseries at Jerilderie and Deniliquin.
However, this visit was not organised as once again the NSW rice industry suffered another season of minimal production (approximately 5% of "normal" production levels), no on-farm trials and significantly reduced resources for the breeding program including staff reductions and a limited number of breeding trials.

The overall aim of the project is to improve and stabilise farmer returns from growing wheat in dry, rainfed environments in NW China by developing higher-yielding wheat germplasm that makes more effective use of water and soil resources.

Objective 1: To develop new germplasm for NW China bred for high transpiration efficiency (TE).
In NW China, the dryland winter-wheat crop relies heavily on effective use of water stored in the deep, loess subsoil. All the water extracted from the subsoil is transpired, so wheat cultivars with high TE should perform well.
During 2008-09, protocols for screening for high TE using the surrogate measure carbon isotope discrimination (CID) were implemented in China following training from the Australian project leader. Genotypic variation in TE within commercially-relevant Chinese germplasm was assessed by measuring CID of Australian varieties and of entries in the crossing blocks of the Chinese partner institutes. Encouragingly useful genotypic variation was identified and this information has been used to develop a strategy for selecting for improved TE within crosses involving Chinese wheats with appropriate agronomic fit. At NWAFU, a porometer was purchased to aid in evaluating alternative strategies of screening for improved TE.

Objective 2: To develop new germplasm for NW China incorporating dwarfing genes sensitive to gibberellic acid (GA) and bred for long coleoptiles.
Long coleoptile length is an important trait in dryland farming because it allows farmers to sow their seed deeper, into moist soil to promote successful crop establishment. The most widely-deployed dwarfing genes strongly limit the growth of the coleoptile because they are GA-insensitive. Alternative, GA-sensitive dwarfing genes are available to facilitate breeding long-coleoptile wheats of agronomically-appropriate height.
During 2008-09, screening of Chinese germplasm for dwarfing-gene status was initiated using (1) CSIRO-developed molecular-markers diagnostic for GA-insensitive dwarfing genes Rht1 and Rht2 and molecular markers for one GA-sensitive dwarfing gene, Rht8, known to be deployed in China, (2) seedling assays of sensitivity to GA and (3) phenotyping of plant height. This information has been used to choose candidate Chinese winter-wheat and spring-wheat parents for use in crosses with alternative dwarfing-gene donors sourced from Australia.
Screening of tall and semi-dwarf Chinese wheats for coleoptile length was also initiated at the partner institutes in NW China.

Objective 3: To develop new germplasm for NW China bred for high early vigour.
The spring-wheat crop in NW China has a very short season and rapid dry matter production should translate into higher grain yield in this short-season wheat environment.
Progress on this objective has been limited to training activities conducted by the Australian project leader and the generation of fresh seed of Chinese germplasm and of germplasm sourced from Australia.

Objective 4: To develop new germplasm for NW China and Australia incorporating root traits for more effective water use.
In NW China, the loess soils are deep and can store large amounts of water. Some of the wheats from this region probably have the capacity for deep root growth that allows them to access more of the soil water store. In this project we are comparing Chinese, Australian and synthetic bread wheats to identify wheats with the deepest root growth for use in crossing.
Screening for root growth using 'cigars' of rolled germination paper, as practiced at CSIRO, has replaced a 'glass-plate' protocol previously used at NWAFU. Screening of Chinese germplasm is revealing useful genotypic variation in root elongation and root number. Both these traits are likely to be important for efficient exploration of the subsoil.

Objective 5: In Australia, to develop breeder-friendly protocols and germplasm that pyramids high leaf transpiration efficiency with greater early vigour.
Computer-simulations indicate that large and consistent yield gains of about 15% are likely in all regions of Australia by pyramiding high TE and greater early vigour together. In project research being conducted in Australia, breeder-friendly protocols are being developed to most-effectively breed and select for this combination of traits.
During 2008-09, new field-based protocols for screening early vigour were tested among progeny of a typical commercial cross in Australia, involving GA-insensitive parents. Techniques using digital photography and spectral reflectance were compared. Initial indications are that both techniques yield similar information, but that digital photography is less-dependent on sunny weather conditions. Evaluation of surrogate measures of TE, to replace the relatively-expensive CID technique, was also initiated. A second, large population of breeding lines varying for TE, vigour and dwarfing-gene type was advanced in the field and evaluated for variation in key agronomic traits of flowering time, height and disease resistance.

The overall aim of the project is to improve and stabilise farmer returns from growing wheat in dry, rainfed environments in NW China by developing higher-yielding wheat germplasm that makes more effective use of water and soil resources.

Objective 1: To develop new germplasm for NW China bred for high transpiration efficiency (TE).
During 2009-10, genotypic variation in TE within commercially-relevant Chinese germplasm was assessed by measuring carbon isotope discrimination (CID) of entries in the crossing blocks of Chinese partner institutes in comparison with CID of Australian varieties sown alongside. This information is being used to implement a strategy for selecting for improved TE within crosses involving Chinese wheats with appropriate agronomic fit. At NWAFU, Shaanxi, and NAAFS, Ningxia, crosses have been made among Chinese parents and also between Chinese and Australian parents. Progeny from these crosses are being advanced for selection and field testing.

Objective 2: To develop new germplasm for NW China incorporating dwarfing genes sensitive to gibberellic acid (GA) and bred for long coleoptiles.
Screening of Chinese germplasm for dwarfing-gene status continued during 2009-10. Information from this screening and from screening in 2008-09 was used to choose candidate Chinese winter-wheat and spring-wheat parents that were used in crosses with alternative dwarfing-gene donors sourced from Australia. Progeny from these crosses are being advanced for selection and field testing.
Screening of tall and semi-dwarf Chinese wheats for coleoptile length was also conducted at the partner institutes in NW China.

Objective 3: To develop new germplasm for NW China bred for high early vigour.
Further training in screening for this trait was conducted by the Australian project leader during the visit to Australia of Chinese project staff in November 2009. Field assessment for early vigour in Chinese germplasm is being conducted on spring-2010 sowings by NAAFS project staff. Data from this assessment will be used to direct a crossing strategy incorporating GA-sensitive dwarfing sources.

Objective 4: To develop new germplasm for NW China and Australia incorporating root traits for more effective water use.
Screening of Chinese germplasm for root growth traits using 'cigars' of rolled germination paper has revealed useful genotypic variation in root elongation and root number. Crosses have been made among Chinese parents and progeny from these crosses are being advanced for selection and field testing.
Dr Zhang Hong, from NWAFU, Shaanxi, returned there in April 2010 after 12 months at CSIRO, Canberra, where he conducted studies on root traits using large breeding populations and mapping populations of wheat derived from Chinese and Australian parents. This work linked closely with work being done under the ACIAR-funded project "Indo-Australia project on root and establishment traits for greater water use efficiency in wheat" (CIM/2006/071). Dr Zhang's research indicated that there are multiple genomic regions associated with important root traits such as root number and root elongation. It was also found that repeatability for root traits was high in genetically fixed lines but moderately low at early generations. This information is being used to direct selection strategies for root traits in this ACIAR project and in project CIM/2006/071.

Objective 5: In Australia, to develop breeder-friendly protocols and germplasm that pyramids high leaf transpiration efficiency with greater early vigour.
During 2009-10 a large population of breeding lines varying for TE, vigour and dwarfing-gene type was sown in replicated field trials in three environments in SE Australia. As well as growth and yield, lines were evaluated for variation in TE and vigour and other key traits such flowering time and height.
Substantial variation was observed among lines in both early vigour and TE, assayed by measuring CID. Above ground biomass at the three leaf stage varied ca. 2.5-fold. Leaf-level TE varied ca. 1.3-fold, a large range for wheat. Early vigour had some dependence on seed size and was also influenced by other factors such as tiller number and leaf width. Greater early vigour was not correlated with leaf thickness. Conversely, an increase in TE tended to involve thicker leaves and an increase in chlorophyll concentration. Early vigour and TE were only weakly related, indicating that they could be selected independently and pyramided in this population.

The overall aim of the project is to improve and stabilise farmer returns from growing wheat in dry, rainfed environments in NW China by developing higher-yielding wheat germplasm that makes more effective use of water and soil resources. Partners in the project are wheat researchers from CSIRO Plant Industry, Canberra and wheat scientists from North-West Agriculture and Forestry University (NWAFU), Yangling, Shaanxi and the Ningxia Academy of Agricultural and Forestry Sciences (NAAFS), Yinchuan and Guyuan, Ningxia.
This report covers activities carried out in the third year of the project. The bulk of these activities involved screening progeny from crosses involving elite Chinese parents for traits identified at CSIRO as important for enhancing yield in dry, rainfed environments. The aim is to advance selected lines for testing in field trials in the next two years of the project.

Objective 1: To develop new germplasm for NW China bred for high transpiration efficiency (TE).
A large number of crosses have been made among Chinese parents differing in TE and also between Chinese and Australian parents. During 2010-11, a small number of key crosses were chosen for agronomic fit and progeny from these crosses sown in short rows in the field. From among these rows, moderately large sets of lines are being selected that are uniform for height and flowering time but which contrast in the expression of TE. The aim during the last two years of the project is to evaluate the impact of selecting for high TE in several field environments in NW China.

Objective 2: To develop new germplasm for NW China incorporating dwarfing genes sensitive to gibberellic acid (GA) and bred for long coleoptiles.
Screening of Chinese germplasm for dwarfing-gene status has confirmed that almost all wheats bred for rainfed production in NW China are either 'tall' wheats or they carry the Rht8 dwarfing gene. This gene has a relatively minor effect on plant height. Alternative dwarfing-gene donors sourced from Australia have been used in crosses and back-crosses with elite Chinese parents. Dwarfing genes Rht4, Rht13, Rht14 and Rht18 have been the main genes targeted. These genes are more effective than Rht8 in reducing plant height but are not extreme. Progeny from these crosses are being advanced for selection and field testing.
Long coleoptiles are a highly-valued character in the dry, rainfed environments of NW China. Screening for coleoptile length among progeny from crosses of Chinese wheats and between Chinese and Australia-sourced wheats continued at the partner institutes in NW China using protocols introduced during the first year of the project.

Objective 3: To develop new germplasm for NW China bred for high early vigour.
Field assessment for early vigour in Chinese germplasm was conducted on spring-2010 sowings by NAAFS project staff. Data from this assessment is being used to direct a crossing strategy to combine early vigour and GA-sensitive dwarfing genes into elite Chinese cultivars. At NWAFU, selections have been made among progeny rows to develop sets of lines contrasting for vigour for field testing during the last two years of the project.

Objective 4: To develop new germplasm for NW China and Australia incorporating root traits for more effective water use.
During the first two years of the project, screening of Chinese germplasm for root growth traits using 'cigars' of rolled germination paper revealed useful genotypic variation in root elongation and root number. Progeny from crosses among elite Chinese parents are now being screened using the 'cigar' technique and also in PVC tubes filled with soil. The latter have been built to specifications obtained during the visit of Chinese scientists to Canberra in November 2009.

Objective 5: In Australia, to develop breeder-friendly protocols and germplasm that pyramids high leaf transpiration efficiency with greater early vigour.
During 2010-11 a large population of breeding lines varying for TE, vigour and dwarfing-gene type was again sown in replicated trials in SE Australia. The extremely wet 2010 season presented difficulties in agronomic management and also interpretation of results for dryland production. Data from 2009 and 2010 trials has been used to truncate the population for more detailed studies in 2011-12.
Glasshouse studies were used to closely investigate the utility of a widely-used leaf chlorophyll meter, the 'SPAD' meter, to assess variation in photosynthetic capacity. High photosynthetic capacity is an important component of high TE, but selection for early vigour may limit photosynthetic capacity and therefore limit TE gain. Some of the observations on the utility of SPAD measurements made in 2010-11 were unexpected and will be pursued further in 2011-12.

The project aim is to increase the water use efficiency and yield of wheat in the rainfed and minimally irrigated regions of Australia and India by developing new breeding lines with deeper root systems that better exploit moisture stored in the soil, and desirable characteristics to enhance crop establishment. A team of physiologists, agronomists and breeders in southern and northern Australia has been assembled to collaborate with a team of leading breeders in India to achieve this aim. Since starting the project on July 1, 2009, the teams have become integrated around a core germplasm collection, common target traits (roots and establishment), common controlled environment and field measurements, and joint data analyses.
The first objective is to identify wheat genotypes with deeper, faster-growing root systems that access more water and result in greater yield. Completed activities include:
(i) sending a diverse collection of novel and high performing germplasm to India for validation within the project (this material will be increased May to September 2010 in northern India for planting in Indian trails in 2010/ 2011 season);
(ii) sharing phenotyping protocols for measuring roots in controlled and field conditions at four face-to-face visits since project inception in 2008 between Australian and Indian researchers, and electronically via a SharePoint site;
(iii) establishing and running field trials for deep root growth at four Australian sites (100 to 400 lines assessed per site) and three Indian sites (40 lines assessed per site) that included water use efficiency and soil moisture measurements, root measurements to depth (by coring) and shoot measurements;
(iv) testing novel methods to indirectly detect wheat lines with deeper root systems in the field using leaf and soil imaging in Australia for use by breeders in both countries in future;
(v) initiating the screening of new lines for faster-growing root systems in controlled conditions in Australia and India for linking to field results; and
(vi) crossing wheat lines with slow and fast root growth to identify genomic regions associated with deeper root systems for marker development in future. Activities in progress towards this objective include receiving Indian germplasm in Australia (MTA being approved and exchanged).
A significant achievement was to measure the maximum rooting depths of wheats at the Indian sites by the PDF on a month-long visit to India in February, to provide a benchmark for improvements with new germplasm in India. This data was not available and is likely to have a wide impact on Indian wheat yield improvements and water use efficiency in future beyond the scope of this project.
The second objective is to develop new germplasm for better crop establishment and robust molecular markers for use in breeding. Activities towards this objective include
(i) sending Australian germplasm with good establishment traits to India for testing in the field in 2011-2012
(ii) training Indian breeders in rapid selection methods (phenotyping) for good establishment traits quickly in the laboratory.
(iii) molecular progress in Australia towards the release of the first marker for Rht13, an alternative dwarfing gene to Rht 1 and 2, which allows wheat to have long coleoptiles but optimal final plant height. A long coleoptile is the key trait to allow deeper seed placement into moisture for better establishment and higher yield in limited moisture environments.
Communication. Project teams communicated face to face on four occasions. In September 2008 three Indian breeders visited Canberra and surrounding field sites for training in techniques to measure root and shoot characteristics; in November 2009 two Australian scientists went to New Delhi for a project meeting; in February 2010 the PDF visited all Indian sites to develop root coring methods and share other field measurements to establish lines with deeper roots and greater water use efficiency. Electronic communication occurs on a SharePoint site established for the project, via email and phone. All groups are trained in common measurement methods, and field data from two Indian seasons at three sites and one Australian season from four sites is being analysed with novel statistical methods and ranking algorithms to identify outstanding lines with superior roots in the field. Project activities were communicated directly to Australian breeding companies at the CSIRO Pre-breeding Information Day for Breeders, in Canberra in September 2009, and to Indian breeders at the first Indo-Australian Program on Marker-Assisted Wheat Breeding (IAP-MAWB) Science Meeting, Australian High Commission, New Delhi in February 2010.
Staffing. The Australian project team hired post-doctoral fellow Dr. Anton Wasson and technician Clare Firth. The Indian project teams hired SRFs in three groups- Pune, Indore and Karnal. This project is on track to meet the objectives and is within budget.

The aim of this project is to develop wheat germplasm for India and Australia with shoot and root traits that enable it to capture more water and convert that water into more grain than current wheat varieties. Australian wheat research groups from CSIRO, Canberra and DEEDI, Queensland, are collaborating with the national Indian wheat breeding organisations, Directorate of Wheat Research (DWR) in Karnal, the Indian Agricultural Research Institute (IARI) in Indore and the ARI in Pune to achieve this aim. The project commenced July 1, 2009. In the first year, phenotyping methods were shared with the Indian groups to select for long coleoptiles to emerge from deep planting in moist soil, and for rapid early leaf vigour to shade soil and minimise early evaporation of soil water. The Indian groups were trained in root phenotyping methods in controlled conditions. Methods were developed in India to core and wash roots in the field, to measure root depth and density and water uptake across many genotypes and sites. Australian germplasm was shipped to India. All Indian and Australian groups carried out field and controlled environment screening for roots of their own country germplasm. This report covers the activities carried out in the second year of the project, from July 1 2010 to June 30 2011.
The first objective is to identify wheat genotypes with deeper, faster-growing root systems that access more water and result in greater yield. Activities this year include:
(i) Germplasm exchange and seed increase
Indian germplasm arrived in Australia under an MTA agreement and is currently being grown in quarantine. This is an important achievement in the project. The material comprises of 40 lines selected by the Indian team representing Indian cultivars with long-running high yields in the central and peninsular water-limiting regions. It was screened over the first three seasons of the project at the three Indian field sites, and this data provides substantial information on its performance and evidence for variation in deep roots and yield. In Australia it will be phenotyped in control and field conditions to compare against Australian germplasm for potential new traits to introgess into Australian varieties. This material took approximately two years to arrive and delayed project activities. Tim Setter and Richard Trethowan provided 10 lines of this set late last year and these are being phenotyped in controlled conditions and the field now, so there is a first look at those lines.
Australian germplasm, which arrived in India in December 2009 under MTA, was increased in the off station nursery over summer 2010 and over the rabi 2010/2011 season at Karnal. Approximately 550 lines yielded an average of 120 g of seed. This is being increased again over the summer 2011 to provide enough seed for all three sites to assess root growth by the lines selected for deeper roots, emergence by the alternative dwarfing gene lines, and ground cover by the leaf vigour lines. Enough seed will be available for some lines to do full plots and assess water use and water use efficiency in India. This will be done following the training scheduled for the groups in India September 2011 with water use efficiency expert John Kirkegaard.
(ii) Field phenotyping of roots in India and Australia
The project team is building a comprehensive, multi-environment data set of wheat root variation for breeding. Field trials for deep effective root growth were run in India with 40 Indian lines at three sites at Karnal, Indore and Pune (third season) and in Australia with 100 Australian lines at four sites (second season). Hill plot configuration and methods developed Year 1 were used. Shoot measurements (green leaf maintenance, canopy temperature depression, harvest index and grain size) were used as indirect measures of root growth and water uptake to develop quick screens for breeders. For the second year, these shoot measures were used to identify lines to core for roots. Root coring to 1.5 m in India was done using the methods developed with the Australian PDF in 2010. Quick shoot measures show good correlations with shoot biomass and harvest index and yield, and weak to good correlations with deep root growth, especially in Indore in India. Analysis is still underway with 2010 and 2011 data. The Australian sites had very high rainfall in 2010 (twice average at grain development in the south; in the north it was too wet to harvest the trials) and data are possibly not relevant to water-limited environments.
(iii) Controlled environment phenotyping of roots
The top 100 Australian lines from the 2009 trials were screened in a controlled environment seedling screen in flat large germination papers to the two leaf stage. Seminal root number, angle and length were measured. The angle that the roots grow from the seed was significantly correlated to root depth in the field; wheats with narrower angles had deeper roots. Root angle varied six-fold across the lines. It may be an important trait to screen for deeper roots in the field, as other groups have suggested. Root number and length at this seedling stage were not correlated with mature plant root depth and length in the field. This confirms previous studies that are suggesting that root length at the seedling stage is not a good predictor of mature root length. However it is a good predictor of seedling length in the field.
Controlled environment phenotyping of roots in India is progressing.
(iv) QTL identification for seedling root traits- collaboration with Tony Condon and the ACIAR-China project
Visiting scientist Dr. Hong Zhang of Northwest A & F University screened a mapped population of recombinant inbred lines (RILs) from a cross between Vigor18 and Chuanmai 18 for QTLs associated with seedling root traits. Several QTL associated with seminal root number, primary root length, total root length, and branching were identified. This population was sent to India and was grown in the field in Australia (2009). Controlled and field phenotyping results will be compared with QTL.
The second objective of the project is to develop new germplasm for better crop establishment and robust molecular markers for use in breeding. Activities towards this objective include:
(i) Marker for Rht 13
Rht 13 remains the preferred alternative dwarfing gene for improved crop establishment (long coleoptile for deep planting into moisture and reduced final plant height). Other Rht genes, and possible markers for them, are also being assessed (Rht 4, 5, 18).
This year a new marker for Rht 13 was identified and found to be informative across 66 Australian and international cultivars. This marker requires capillary electrophoresis for discrimination, a method not readily available for breeders. Efforts are underway to get closer to the gene to develop a gel based assay for breeders. This coming year field research will test whether there is any Rht gene by drought interactions, and this will be conducted at the three Indian sites with germplasm with these genes.
Mentoring and training.
Project members in Australia are working with ACIAR Graduate Officer Ms. Keshia Hilliam to foster a stronger community around early career researchers in wheat and agriculture within this project and the Indo-Australian Program on Marker-Assisted Wheat Breeding (IAP-MAWB).
Communication.
(i) In India
Project teams travelled together to the three Indian sites, Karnal, Indore and Pune in February 2011, to further develop root coring methods and share other field measurements to establish lines with deeper roots and greater water use efficiency. This project tour followed the second Indo-Australian Program on Marker-Assisted Wheat Breeding (IAP-MAWB) Science Meeting, Directorate of Wheat Research, Karnal in February 2010. Electronic communication occurs on via email, Skype and phone.
(ii) In Australia
Project presented to the ACIAR-India Minisymposium in Adelaide, October 2010.
(iii) Publications
An abstract was jointly written and submitted to the International Botanical Congress in Melbourne 2011 to be followed by a publication in the Journal of Experimental Botany September 2011.
Media coverage in Ground Cover, Partners magazine and journal Nature.
Staffing.
The Australian project team hired technician Samantha Walker to replace Clare Firth, and she joins PDF Anton Wasson. The Indian project teams have SRFs in three groups- Pune, Indore and Karnal.
Delays.
This project is approximately one year behind towards meeting the objectives due to delay in exchange and bulking of germplasm in each country and an extraordinarily wet season in Australia in 2010. It is within budget.

A robust molecular marker for the stem rust resistance gene, Sr22, effective against the Ug99 strain and derived lineages as well as Indian and Australian isolates was tested on a wide range of Indian and Australian wheat genotypes being used in cultivar development. The marker previously described in the SRA preceding the current project, proved to be highly diagnostic for the presence or absence of the Sr22 resistance. Following on from validating the utility of the marker, the information required for utilisation in "marker assisted wheat breeding" has been disseminated to participating scientists/institutions in the India-Australia wheat improvement program.
As part of the ongoing effort to identify additional stem rust resistance genes that interact with the durable adult plant leaf and stripe rust resistance gene (Lr34/Yr18) to provide enhanced stem rust resistance against Ug99 and other strains, F2 families derived from RL6058 and Chris were scored for stem rust resistance at Cobbitty in the 2009 field season. The wheat genotypes, Chris and RL6058, carry Lr34/Yr18 and an unknown number of stem rust resistance genes. To ensure reliable stem rust infection and rust scores, the F3 progeny will be tested in Wellington (India) and Cobbitty (Australia) in 2010. Preliminary efforts to identify the unknown stem rust resistance genes using molecular marker-genetic linkage analysis pointed to a region on wheat chromosome 2B as harbouring the stem rust resistance gene(s).
Objective 3 of the project was aimed at making comparisons of variability in the stem rust pathogen between India and Australia. These studies are expected to contribute to the overall project aim of developing more durable resistance to stem rust by improving our ability to pre-empt pathogen change by gaining a deeper understanding of how P. graminis f. sp. tritici (Pgt) generates genetic variability. Discussions were held with Dr Mohinder Prashar (Directorate of Wheat Research Regional Station at Flowerdale) in India during March 2010 on the methods used in wheat stem rust surveillance and identification of pathotypes of stem rust in India and Australia. Although delays to the start of the project slowed progress in some activities, planning is well advanced and at this stage, no foreseeable problems are expected in delivering the contracted milestones under objective 3 by project completion.

Two types of stem rust resistance to the Ug99 stem rust pathotype and other isolates from India and Australia are investigated as through the ACIAR-ICAR project. The resistance genes Sr13 and Sr22 are of the seedling resistance category. We previously reported on a diagnostic marker for Sr22 distributed to Indian and Australian wheat breeding programs. In keeping with a commitment to the wider wheat rust research community to ensure that the type and source of resistance in cultivars and breeding materials are well characterised the marker information from the ACIAR project has now been disseminated widely through publication in an international journal.
The other category of stem rust resistance is the post seedling resistance commonly referred to as adult plant resistance (APR). New sources of stem rust APR continues to be the focus of the project using germplasm collected in the early 1900's, which are referred to as the Watkins collection. Two wheat genotypes AUS28082 and AUS27856 from the collection were established to carry adult plant stem rust resistance. However, inheritance of the APR is not known. F3 populations from two crosses AUS28082/Yitpi and AUS27856/Yitpi were screened during the cropping season in 2009. Marker genotyping using known APR genes, Sr2 and Lr34-linked markers, suggested that these genotypes carry adult plant resistance gene(s) different to these known genes. Two F3 families showing putatively single gene segregation from the respective population were identified. Up to 200 F3 seeds of each of these families were space-planted to generate F3 equivalent of monogenically segregating populations (MSPs) during the crop season 2010. These progenies will be screened against stem rust at Cobbitty during the 2011 cropping season. Phenotypic results will be used to pool DNA samples from putative homozygous resistant and susceptible plants to determine chromosomal location of APR to stem rust. AUS28082/Yitpi-derived F5 population will be planted in Wellington, India and Cobbitty Australia during 2011 crop season.
Another component of the APR work is based on additional stem rust resistance genes that interact with the durable adult plant leaf and stripe rust resistance gene (Lr34/Yr18) to provide enhanced stem rust resistance against Ug99, found in wheat genotypes such as Chris and the Thatcher derivative, RL6058. Field trials in 2010 at Cobbitty and Wellington in India failed to provide reliable stem rust scores for APR in RL6058 and Chris crosses. Some of the lines showed differences in flowering time and appeared to be a confounding factor in the stem rust phenotype evaluations. We selected a subset with similar flowering time for subsequent rust phenotyping. We are phenotyping lines in three locations (Kenya, Minnesota and Australia) in 2011 to ensure that we receive reliable rust scores from at least one of these locations.
A region on wheat chromosome 2B that is linked to stem rust resistance was identified in Thatcher background and more SSR markers were mapped to the linkage group. We also identified additional SSRs which are polymorphic between parental lines to help facilitate the process of identifying other genomic regions involved in the improved stem rust resistance..

Objectives

Objective 1 (Yr 1, m1 to Yr 3, m12)
Fertilization-independent (FI) formation of rice endosperm and pericarp
Output 1.1 Refined FI pericarp formation based on understanding of OsAsp1 function (I)
Output 1.2 Refined FI endosperm formation based on RNAi of OsFIS genes (C)
Output 1.3 Line combining FI pericarp formation and FI endosperm formation (I + C)
Objective 2 (Yr 1, m1 to Yr 3, m12)
FI embryogenesis in rice nucellus
Output 2.1 Isolated rice orthologue of maize Mac1 gene (I)
Output 2.2 Isolated rice orthologues of Arabidopsis CLV, WUS, STM and AG genes (C + I)
Output 2.3 System to control genes of nucellus from megaspore mother cell (MMC1) (I)
Output 2.4 OsMac1-based system for inducing second MMC (MMC2) (I)
Output 2.5 WUS-induced FI embryogenesis in MMC2 (C +I)
Output 2.6 WUS-induced FI embryogenesis in nucellar cluster proximal to MMC1 (C)
Objective 3 (Yr 4, m1 to Yr 5, m12)
Apomictic hybrid rice
Output 3.1 Line displaying FI formation of embryo, endosperm and pericarp (I + C)
Output 3.1 Line in which endosperm feeds apomictic embryo during germination (I + C)
Output 3.3 One-line apomictic hybrid rice (I + C)
Objective 4 (Yr 1, m1 to Yr 5, m12)
Communication and dissemination of research results
Output 4.1 Publications and website for pre-publication releases (Yr 1, m3)
Output 4.2 Participation in Third International Symposium on Apomixis (Yr.3, m?)
Output 4.3 Mid-term review at CSIRO (Yr 3, m10)
Output 4.4 Participation in Fourth International Congress on Hybrid Rice (Yr 4, m?)
Output 4.5 Terminal workshop at IRRI (Yr 5, m10)

Note: Only those outputs scheduled for initiation in Year 1 are mentioned.
Note: (I) = IRRI activity, (C) = CSIRO activity

Objective 1 (7/'03-6/'06)
Fertilization-independent (FI) formation of rice endosperm and pericarp
Output 1.1 Refined FI pericarp formation based on understanding of OsAsp1 function (I)
OsAsp1 encodes a putative aspartate proteinase that is unusual among plant aspartate proteinases in lacking the Plant Specific Insert. When an OsAsp1:gus promoter:reporter gene fusion was transferred into rice using Agrobacterium, a form of parthenogenesis was observed (FI expansion of the pericarp and seed coat, with a clear aqueous solution instead of embryo and endosperm). This ability to by-pass the checkpoint that normally prevents ovary expansion in the absence of fertilization may be very important in relation to achieving full FI endosperm expansion (see Outputs 1.2 and 1.3). We have now shown that recombinant OsAsp1 expressed in E. coli does indeed encode an active proteinase, which is capable of not only hydrolyzing artificial substrates but also removing its own N-terminal pro-sequence.
Output 1.2 Refined FI endosperm formation based on RNA-I of OsFIS genes (C)
Mutations in Arabidopsis FIS genes produce the FI seed phenotype. We introduced an RNA-I against a rice gene (FIS3 from rice, or FIR) homologous to Arabidopsis FIS3 gene. Transgenic lines harbouring the FIR-RNA-I contained shrivelled grains, similar to fertilized fis3 mutant lines. Autonomous grain-like structures can be seen in the transgenic lines amongst empty grains at a 1:1 ratio. More work is needed in the FIR-RNA-I line to actually demonstrate autonomous growth of endosperm in these lines. The observation of empty pericarp in FIR-RNA-I lines could have implications for Output 1.3. Similar to work was reported from IRRI earlier (Output 1.1). We also observed FI pericarp structure in a tagged line isolated in CSIRO by Dr. Narayana Upadhyaya and colleagues. Cloning DNA adjacent to the insertion site identifies the gene as a small dehydrogenase/hydrolase in Chromosome 9. In Arabidopsis we showed that an antisense construct against the DNA methyltransferase (MET) gene causes FI empty seed formation. We have made RNA-I against the rice MET1 gene and the construct is about to be introduced into rice. Recent sequencing data from rice also indicate that there are 2 FIS3 like genes in rice.

Objective 2 (7/'03-6/'06)
FI embryogenesis in rice nucellus
Output 2..1 Isolated rice orthologue of maize Mac1 gene (I)
This work was accomplished by the Kurata labortatory of the National Institute of Genetics in Mishima, Japan. See Nonomura et al. (2003) Plant Cell. 15:1728-1739. As predicted in the Phase 1 grant proposal, Msp1, the rice orthologue of maize Mac1, is a leucine-rich repeat receptor-like protein kinase.
Output 2..2 Isolated rice orthologues of Arabidopsis CLV, WUS, STM and AG genes (C + I)
Putative rice orthologues of WUS and AG have been identified at IRRI. Their functional identities are now being confirmed. STM and AMP1 homologues have been identified are being isolated in Canberra.
Output 2..3 System to control genes of nucellus from megaspore mother cell (MMC1) (I)
We have identified 2 rice genes that are putative ligands of Msp1 and that may control the behavior of surrounding nucellar cells through interaction with Msp1. RNA in situ hybridization and RNA-I are being used to confirm and characterize the putative ligands. These genes will be tested for their ability to constitute a system for controlling gene expression in nucellar cells from the MMC. If successful, the system will be used to drive nucellar embryogenesis at the time of meiosis.
Output 2..4 OsMac1-based system for inducing second MMC (MMC2) (I)
The phenotype of Tos17 knock-outs of Msp1 is multiple sporocytes, including multiple MMCs. This is a good start towards generating just one additional MMC (MMC2), but we need to exert finer control than is possible with knock-outs. The system discussed under Output 2..3 will be modified to achieve this result.

Objective 4 (7/'03-6/'08)
Communication and dissemination of research results
Output 4.1 Publications and website for pre-publication releases (10/'03)
This activity has been postponed until 1/'05 because of the need to complete publication of Phase 1 data and obtain approval for release of Phase 2 data that are currently confidential.

[Note: Only outputs scheduled for initiation in Years 1-3 are mentioned. Activities: (I) = IRRI, (C) =
CSIRO.]
Our goal is to make the benefits of hybrid rice available to poor farmers through asexual seed
production. At present, farmers must buy expensive hybrid seed fresh every season, because its
heterozygosity is diminished by 50% with every cycle of sexual reproduction. We propose to develop
a synthetic form of apomixis that will fix the heterozygosity of the hybrid and allow farmers to
reproduce hybrid seeds cheaply in their own fields. Although essentially all natural apomicts are
polyploids, we expect our synthetic apomixis to be compatible with diploidy in rice.
We shall model our synthetic apomixis on the apospory of Poa pratensis, a relative of wheat and
barley in which only 5 genetic loci are required for apospory [Matzk et al. (2005) Plant Cell 17:13]24].
The key milestone will be the generation of an aposporous embryo from diploid cells in the nucellus
of the ovary. We shall convert 1-2 cells of the nucellus of rice into the equivalent of aposporous initials
(AIs) and assess their capacity to develop firstly into diploid embryo sacs and then into aposporous
embryos.
The conversion of nucellar cells into aposporous initials will take advantage of a recently
discovered gene (MSP1) that limits the number of nucellar cells able to become megaspore mother
cells (MMCs). This gene was inactivated by Tos17 insertional mutation. When the mutant was
recovered in the homozygous form (msp1 msp1), up to 15 secondary MMCs form in the ovule and
develop into haploid embryo sacs. We propose to inactivate MSP1 more subtly by RNA interference
(RNAi) to allow only 1-2 secondary MMCs to form. At the same time we shall prevent these cells from
entering meiosis, so that they form the equivalent of diploid AIs. We shall assess the conditions under
which the putative AIs form diploid embryo sacs and aposporous embryos. Objective 2 focuses on
generating an apomictic embryo by the above approach.
Apomictic embryogenesis may be pursued with or without fertilization. The fertilizationdependent
approach is in some respects more straightforward because formation of the endosperm
normally requires fertilization, even in many apomicts, and the seed coat and the pericarp, although
maternal tissues, usually fail to develop fully without fertilization. Furthermore, the fertilizationdependent
approach would produce the usual triploid endosperm rather than a diploid form. We
have opted here for the fertilization]independent (FI) approach because (1) it eliminates competition
between the sexual embryo and the apomictic embryo, and (2) it avoids controversy about the escape
of apomixis genes into the environment. However, it will be necessary to have a transient sexual
phase for the initial formation of the apomictic hybrids in breedersf fields; the FI phase can be used in
farmersf fields. Objective 1 focuses on the FI protocol.
Objective 1 (7/ 03-6/ 06)
Fertilization]independent (FI) formation of rice endosperm and pericarp
.. Output 1.1 Refined FI pericarp formation based on understanding of OsAsp1 function (I)
Both IRRI and CSIRO have engineered methods for by]passing the fertilization]dependence of
pericarp and seed coat enlargement. This by-pass is essential for achieving FI-independent
seed production, because it removes a physical constraint on the size of the endosperm that
can develop autonomously.
.. Output 1.2 Refined FI endosperm formation based on RNA]I of OsFIS genes (C)
FI endosperm formation in rice is essential for constructing a form of apomixis in which the
escape of transgenic pollen is prevented. CSIRO has a method of generating fertilisationindependent
formation of the endosperm in Arabidopsis, based on the discovery through
mutation of three Fertilization-Independent Seed (FIS) genes. This method has been introduced
into rice. Based on sequence similarity, all the FIS like genes in rice had been identified, and
transgenic lines with silencing constructs for each OsFIS gene are being evaluated.

Objective 2 (7/ 03-6/ 06)
FI embryogenesis in rice nucellus

.. Output 2.1 Isolated rice orthologue of maize MAC1 gene (I)
We are focusing on developing a synthetic form of apospory for rice. Aposporous embryos
develop from nucellar cells near the megaspore mother cell (MMC). The maize mutation
multiple archaesporial cells1 (mac1) showed that such nucellar neighbours can develop into
secondary MMCs if the MAC1 gene is inactivated. One of IRRIfs objectives was to clone the
rice orthologue, OsMAC1. However, this task was actually accomplished by Nonomura et al.
(2003) in Japan [Plant Cell. 15:1728]1739], and they named the rice gene MULTIPLE
SPOROCYTES1 (MSP1).
To identify the MSP1 promoter, Ms Xinai Zhao of IRRI examined the region 5 kb upstream
from the MSP1 transcription start site defined by Nonomura et al. (2003). She found that the
nearest upstream gene, which had been predicted by annotation software to be transcribed in
the opposite direction from MSP1, was in fact transcribed in the same direction as MSP1. As
the distance between the two genes was very small, this finding raised the possibility that the
upstream gene was really part of the MSP1 gene; either the transcriptional start site for MSP1
might be further upstream than previously thought, or there might be multiple start sites. Ms
Zhao has now assembled considerable data to support the idea that the MSP1 gene has a very
long 5f]untranslated region. She is currently conducting RNA gel blots and complementation
studies of the homozygous msp1 mutant to identify the promoter and use it in RNAi to control
MSP1 transcript levels.
A key paper on the genetics of the control of apomixis and sexuality in Poa pratensis was
published by Matzk et al. (2005) Plant Cell 17:13-24. In this paper, the authors identified a
locus APV (Apospory Prevention) and speculated that it may be the Poa ortholog of MAC1.
They identified two other loci governing the initiation of apospory (AIT, Apospory Initiation,
and mdv, Megaspore Development) controlling apomeiosis and the initiation of the sexual
pathway. IRRI is studying candidate genes as orthologs of these loci. Matzk et al. (2005)
identified two loci controlling parthenogenesis in Poa, ppv (parthenogenesis prevention) and
PPI (Parthenogenesis Initiation). In Arabidopsis, a newly identified Polycomb protein that
interacts with FIS complex, AtMSI1 may code for ppv (see section 4.2 for details). CSIRO
isolated the OsMSI1 and testing the alternative way to induce parthenogenesis in rice.

.. Output 2.2 Isolated rice orthologues of Arabidopsis CLV, WUS, STM and AG genes (C & I)
Embryogenesis in the rice nucellus will probably require re-activation of meristematic activity
through expression of orthologues of such Arabidopsis genes as CLAVATA1]3, WUSCHEL,
SHOOT MERISTEMLESS and AGAMOUS. IRRI and CSIRO identified orthologues by
sequence comparisons. In the last year, Mr. Rico Gamuyao of IRRI examined 10 members of
the rice WOX gene family to ascertain their expression patterns, using reverse transcriptionpolymerase
chain reaction (RT-PCR) and RNA in situ hybridization. He found that two genes
were highly expressed in the ovule. One of these two genes, the one most closely related in
sequence to WOX11 and WOX12 of Arabidopsis, was highly expressed also in anthers, root
tips and the shoots of 2-day-old germinated seeds. The other gene, most closely related in
sequence to WOX9 of Arabidopsis, was highly expressed only in the ovule (nucellus and
integuments) but was apparently not expressed in the MMC. In Arabidopsis, WOX9 appears
to act by maintaining cell division and preventing premature differentiation [Wu et al. (2005)
Curr Biol 15: 436-]440]. We are studying whether the rice gene has a similar function
principally in the ovule.

.. Output 2.3 System to control genes of nucellus from megaspore mother cell (MMC1) (I)
Leucine]rich receptor kinases like MSP1 are usually triggered by the binding of extracellular
ligands to the LRR domain. We are attempting to identify the ligand of MSP1 and its site of
synthesis. We are guided in this search by the finding that a LRR receptor kinase is implicated
in the control of tapetum development by pollen mother cells, PMC, in Arabidopsis. EXCESS
MALE SPOROCYTES1 (EMS1, also known as EXS) encodes a LRR receptor kinase and is
expressed in the tapetum. TAPETUM DETERMINANT1 (TPD1) encodes a small protein and is
expressed in PMCs [Yang et al. (2005) Plant Physiol. 139:186-191, Yang et al. (2003) Plant Cell
15:2792-2804]. It is possible that TPD1 interacts with the LRR domain of EMS1/EXS, possibly
disrupting a further interaction with a second LRR receptor kinase, SERK1/2 [Colcombet et al.
(2005) Plant Cell 17: 3350- 3361; Albrecht et al. (2005) Plant Cell 17: 3337-3349]. IRRI is
examining the hypothesis that rice contains a gene similar to TPD1. Two putative orthologs
(OsTPD1A and OsTPD1B) have been identified and RNA in situ hybridization indicates that
they are overlap with MSP1 in their sites of expression in the anthers, as shown already for
TPD1 and EMS1/EXS. In ovules, MSP1 and OsTPD1A are expressed. Anthers of homozygous
msp1 mutants have a layer of secondary meiocytes in stead of a tapetum. In such anthers, only
OsTPD1A is highly expressed, with transcripts of MSP1 and OsTPD1B not readily detected.
IRRI and CSIRO are collaborating to use the two]hybrid system of yeast to determine whether
OsTPD1 or OsTPD2 binds to the LRR domain of MSP1.

.. Output 2.4 OsMac1]based system for inducing secondary MMC (MMC2) (I)
In the homozygous msp1 mutant, up to 15 secondary MMCs are induced in each ovule. IRRI is
now attempting to interfere with the function of MSP1 more subtly to induce only 1-2
secondary MMCs which will then be induced to form aposporous initials through the bypassing
of meiosis. Two methods of interfering with MSP1 function are being developed: (1)
subtle down-regulation of MSP1 gene expression through RNAi under the control of the
MSP1 promoter, and (2) subtle down]regulation of the gene encoding ligand of MSP1
(putatively OsTPD1A or OsTPD1B) by RNAi. Two transgenic plants designed for RNAi of
OsTPD1A have been produced at IRRI by Ms Justina de Palma and Ms Gina Borja in the
laboratory of Dr. Philippe Herve. These plants have already been shown by reverse
transcription-PCR to down]regulate OsTPD1A expression by several fold. We are currently
examining these plants for the desired phenotype, namely, the presence of 1-2 secondary
MMCs in the ovule.

Objective 4 (7/f03]6/f08)
Communication and dissemination of research results
.. Output 4.1 Publications and website for pre]publication releases (10/f03)
Publication
Bi X, Khush GS, Bennett J. 2005. The rice nucellin gene ortholog OsAsp1 encodes an active
aspartic protease without a plant]specific insert and is strongly expressed in early embryo.
Plant Cell Physiol. 46:87-98.
Website
A website for the project is being constructed. It will contain information on Phase 1 and
Phase 2, including publications and reports.

[Note: Only outputs scheduled for initiation in Years 1-3 are mentioned. Activities: (I) = IRRI, (C) = CSIRO.]

Our goal is to make the benefits of hybrid rice available to poor farmers through asexual seed production. At present, farmers must buy expensive hybrid seed fresh every season, because its heterozygosity is diminished by 50% with every cycle of sexual reproduction. We propose to develop a synthetic form of apomixis that will fix the heterozygosity of the hybrid and allow farmers to reproduce hybrid seeds cheaply in their own fields. Although essentially all natural apomicts are polyploids, we expect our synthetic apomixis to be compatible with diploidy in rice.
We shall model our synthetic apomixis on the apospory of Poa pratensis, a relative of wheat and barley in which only 5 genetic loci are required for apospory [Matzk et al. (2005) Plant Cell 17:13-24]. The key milestone will be the generation of an aposporous embryo from diploid cells in the nucellus of the ovary. We shall convert 1-2 cells of the nucellus of rice into the equivalent of aposporous initials (AIs) and assess their capacity to develop firstly into diploid embryo sacs and then into aposporous embryos.
The conversion of nucellar cells into aposporous initials will take advantage of a recently discovered gene (MSP1) that limits the number of nucellar cells able to become megaspore mother cells (MMCs). This gene was inactivated by Tos17 insertional mutation. When the mutant was recovered in the homozygous form (msp1 msp1), up to 15 secondary MMCs form in the ovule and develop into haploid embryo sacs. We propose to inactivate MSP1 more subtly by RNA interference (RNAi) to allow only 1-2 secondary MMCs to form. At the same time we shall prevent these cells from entering meiosis, so that they form the equivalent of diploid AIs. We shall assess the conditions under which the putative AIs form diploid embryo sacs and aposporous embryos. Objective 2 focuses on generating an apomictic embryo by the above approach.
Apomictic embryogenesis may be pursued with or without fertilization. The fertilization-dependent approach is in some respects more straightforward because formation of the endosperm normally requires fertilization, even in many apomicts, and the seed coat and the pericarp, although maternal tissues, usually fail to develop fully without fertilization. Furthermore, the fertilization-dependent approach would produce the usual triploid endosperm rather than a diploid form. We have opted here for the fertilization-independent (FI) approach because (1) it eliminates competition between the sexual embryo and the apomictic embryo, and (2) it avoids controversy about the escape of apomixis genes into the environment. However, it will be necessary to have a transient sexual phase for the initial formation of the apomictic hybrids in breeders' fields; the FI phase can be used in farmers' fields. Objective 1 focuses on the FI protocol.

Objective 1 (7/'03-6/'06)
Fertilization-independent (FI) formation of rice endosperm and pericarp
Output 1.1 Refined FI pericarp formation based on understanding of OsAsp1 function (I)
Both IRRI and CSIRO have engineered methods for by-passing the fertilization-dependence of pericarp and seed coat enlargement. This by-pass is essential for achieving FI-independent seed production, because it removes a physical constraint on the size of the endosperm that can develop autonomously.
Output 1.2 Refined FI endosperm formation based on RNA-I of OsFIS genes (C)
FI endosperm formation in rice is essential for constructing a form of apomixis in which the escape of transgenic pollen is prevented. CSIRO has a method of generating fertilisation-independent formation of the endosperm in Arabidopsis, based on the discovery through mutation of three Fertilization-Independent Seed (FIS) genes. This method has been introduced into rice. Based on sequence similarity, all the FIS like genes in rice had been identified, and transgenic lines with silencing constructs for each OsFIS gene are being evaluated.

Objective 2 (7/'03-6/'06)
FI embryogenesis in rice nucellus
Output 2.1 Isolated rice orthologue of maize MAC1 gene (I)
We are focusing on developing a synthetic form of apospory for rice. Aposporous embryos develop from nucellar cells near the megaspore mother cell (MMC). The maize mutation multiple archaesporial cells1 (mac1) showed that such nucellar neighbours can develop into secondary MMCs if the MAC1 gene is inactivated. One of IRRI's objectives was to clone the rice orthologue, OsMAC1. However, this task was actually accomplished by Nonomura et al. (2003) in Japan [Plant Cell. 15:1728-1739], and they named the rice gene MULTIPLE SPOROCYTES1 (MSP1).
A key paper on the genetics of the control of apomixis and sexuality in Poa pratensis was published by Matzk et al. (2005) Plant Cell 17:13-24. In this paper, the authors identified a locus APV (Apospory Prevention) and speculated that it may be the Poa ortholog of MAC1. They identified two other loci governing the initiation of apospory (AIT, Apospory Initiation, and mdv, Megaspore Development) controlling apomeiosis and the initiation of the sexual pathway. IRRI is studying candidate genes as orthologs of these loci. Matzk et al. (2005) identified two loci controlling parthenogenesis in Poa, ppv (parthenogenesis prevention) and PPI (Parthenogenesis Initiation). In Arabidopsis, a newly identified Polycomb protein that interacts with FIS complex, AtMSI1 may code for ppv (see section 4.2 for details). CSIRO isolated the OsMSI1 and testing the alternative way to induce parthenogenesis in rice.
Output 2.2 Isolated rice orthologues of Arabidopsis CLV, WUS, STM and AG genes (C + I)
Embryogenesis in the rice nucellus will probably require re-activation of meristematic activity through expression of orthologues of such Arabidopsis genes as CLAVATA1-3, WUSCHEL, SHOOT MERISTEMLESS and AGAMOUS. IRRI and CSIRO identified orthologues by sequence comparisons. In the last year, Mr. Rico Gamuyao of IRRI examined 10 members of the rice WOX gene family to ascertain their expression patterns, using reverse transcription-polymerase chain reaction (RT-PCR) and RNA in situ hybridization. He found that two genes were highly expressed in the ovule.
Output 2.3 System to control genes of nucellus from megaspore mother cell (MMC1) (I)
Leucine-rich receptor kinases like MSP1 are usually triggered by the binding of extracellular ligands to the LRR domain. We are attempting to identify the ligand of MSP1 and its site of synthesis. We are guided in this search by the finding that a LRR receptor kinase is implicated in the control of tapetum development by pollen mother cells, PMC, in Arabidopsis. EXCESS MALE SPOROCYTES1 (EMS1, also known as EXS) encodes a LRR receptor kinase and is expressed in the tapetum. TAPETUM DETERMINANT1 (TPD1) encodes a small protein and is expressed in PMCs [Yang et al. (2005) Plant Physiol. 139:186-191, Yang et al. (2003) Plant Cell 15:2792-2804]. It is possible that TPD1 interacts with the LRR domain of EMS1/EXS, possibly disrupting a further interaction with a second LRR receptor kinase, SERK1/2 [Colcombet et al. (2005) Plant Cell 17: 3350 - 3361;
Albrecht et al. (2005) Plant Cell 17: 3337 - 3349]. IRRI is examining the hypothesis that rice contains a gene similar to TPD1.
Output 2.4 OsMac1-based system for inducing secondary MMC (MMC2) (I)
In the homozygous msp1 mutant, up to 15 secondary MMCs are induced in each ovule. IRRI is now attempting to interfere with the function of MSP1 more subtly to induce only 1-2 secondary MMCs which will then be induced to form aposporous initials through the by-passing of meiosis. Two methods of interfering with MSP1 function are being developed: (1) subtle down-regulation of MSP1 gene expression through RNAi under the control of the MSP1 promoter, and (2) subtle down-regulation of the gene encoding ligand of MSP1.

Our goal is to make the benefits of hybrid rice available to poor farmers by developing a cheaper and more flexible form of hybrid seed production that allows farmers to multiply seeds in their own fields. We aim to fix the heterozygosity of hybrids through a form of apomixis - seed production in which the genetic constitution of progeny plants is identical to that of the hybrid. To maintain a constant genetic constitution, the embryo of the progeny seed must be formed without undergoing meiosis, a form of sexual recombination. The form of apomixis that we plan to introduce into rice is known as apospory and exists in many wild grasses but not in rice or its close relatives.
In wild grasses, apospory develops in cells alongside the megaspore mother cell (MMC) of the rice ovule. These cells (aposporous initials, AI) are similar to the MMC but differ from it in failing to undergo meiosis. Multiple secondary MMCs are observed in the ovules of the mac1 mutant of maize and the msp1 mutant of rice. We hypothesized that these secondary MMCs may be a first step towards apospory and that the next step would be to by-pass meiosis in the secondary MMCs.
Unfortunately, the msp1 mutant is not a convenient platform from which to develop AIs in rice because it is male-sterile. Staff of IRRI searched for a rice gene other than MSP1 that could be inactivated to produce secondary MMCs in the ovule without causing male sterility. Based on studies in Arabidopsis thaliana, we suspected that MSP1 protein interacts with a small protein that we term TPD1-like1, or TDL1. We found two close homologues of TPD1 in the rice genome (TDL1A and TDL1B). Furthermore, these two genes are co-expressed during meiosis with MSP1. We used yeast 2-hybrid analysis and bimolecular fluorescence complementation to show that TDL1A is a ligand of MSP1. Furthermore, when we used RNA interference (RNAi) to down-regulate the OsTDL1A gene, we found that the OsTDL1A-RNAi transgenic plants produce secondary MMCs in the ovule without causing male sterility. This work has been submitted to Plant Journal for publication. We are now developing a method for by-passing meiosis in the secondary MMCs to produce AIs in rice.
Naturally occurring AIs form embryo sacs that undergo parthenogenesis to produce embryos without fertilization. Again, studies on Arabidopsis thaliana suggested that inactivation of the polycomb complex is essential not only for parthenogenesis but also for autonomous endosperm formation. Staff of CSIRO Plant Industry have, accordingly, isolated the genes encoding the rice polycomb complex. We used either RNAi or insertion mutagenesis to interfere with the expression of these genes and found that two distinct checkpoints in the sexual development of the ovary have been overcome: (i) the checkpoint that prevents ovary enlargement in the absence of fertilization and (ii) the checkpoint that prevents endosperm formation without fertilization of the cell central of the embryo sac. The rice homologue of the Arabidopsis gene CLF appears to be especially important in this context. The next steps are (i) to combine these two features to obtain large autonomous endosperms and then (ii) to manipulate the polycomb complex further to obtain parthenogenetic embryo formation. For step (ii), we are exploring the rice homologues of the Arabidopsis gene MSL1, which mediates signalling between endosperm and embryo.
In summary, our data support the emerging consensus that the sexual and aposporous pathways of plant seed production share many genes but differ at two major stages: (i) the onset of meiosis and (ii) the checkpoints preventing seed development in the absence of fertilization. This consensus raises the intriguing evolutionary question as to whether sexuality is a modification of apomixis or vice-versa.

In July 1997, CSIRO and IRRI began a three-phase project to develop apomixis for hybrid rice (Oryza sativa L.), funded by ACIAR. The goal was to make the yield advantage and stress tolerance of hybrid rice available to poor farmers in Asia and Africa by using synthetic apomixis to reduce the cost and increase the flexibility of hybrid seed production.

Phase 1 (July 1997-June 2002) involved three laboratories: CSIRO Horticulture in Adelaide, CSIRO Plant Industry in Black Mountain, and IRRI in the Philippines. It provided new insights into the similarities and differences between sexual reproduction and asexual apomixis. Highlights were (i) the identification of three FERTILIZATION-INDEPENDENT SEED (FIS) genes in the sexual plant Arabidopsis that form part of a complex to repress endosperm formation in the absence of fertilization and (ii) confirmation that FIS homologues exist in rice and in the natural apomict Hieracium piloselloides that reproduces by apospory (avoidance of meiosis).

Phase 2 (July 2003-June 2008) focused exclusively on rice and involved the Black Mountain Laboratory and IRRI. The three main objectives for Phase 2 were as follows:

Objective 1: To identify all of the known FIS orthologues in the rice genome and to functionally test each for efficacy in fertilization-independent (FI) formation of the pericarp and seed coat of rice (Objective 1a) and FI formation of the embryo and the endosperm in the rice ovule (Objective 1b).
Objective 2: To develop FI embryos in the nucellus by inducing aposporous initials (AIs).
Objective 3: To combine the above traits to generate a basic form of apospory (meiotic avoidance) with embryo and endosperm induction in rice that could be refined further in Phase 3.

Both IRRI and CSIRO contributed to Objective 1a. They found that the checkpoint that normally prevents FI formation of the pericarp and seed coat could be bypassed using transgenic approaches. However, progress with these approaches was hampered by a background level of ovary enlargement in non-transgenic control plants. A change in rice variety from Nipponbare to one in which this background is negligible would be valuable. Objective 1b was pursued by CSIRO. Based on sequence similarity with the Arabidopsis FIS gene, seven candidate rice FIS-like (OsFIS) genes were identified in the rice genome.

Transgenic rice lines containing silencing constructs to down-regulate all of these OsFIS genes were generated and evaluated, and T-DNA insertion lines in three of the FIS-like rice genes were also evaluated. Unexpectedly, autonomous endosperm formation and autonomous embryo formation were not observed in any of the hundreds of transgenic rice lines analyzed. Data on expression of these genes, combined with an evolutionary analysis of the rice and Arabidopsis genes, indicated that rice FIS-like genes do not function in an equivalent manner to that found in Arabidopsis and its relatives. Repression of rice seed formation is likely to occur by an as yet unknown mechanism that needs further investigation if synthesis of apomixis is to be attempted (Luo et al, submitted).

Under Objective 2, the first milestone was to induce the formation of multiple secondary megaspore mother cells (MeMCs) in the nucellus. This milestone was reached (Zhao et al 2008) by silencing OsTDL1A, the rice orthologue of the Arabidopsis gene TAPETUM DETERMINANT1. IRRI showed that OsTDL1A-silenced lines produce secondary MeMCs in both indica and japonica genetic backgrounds. The second milestone is to convert secondary MeMCs into AIs by eliminating meiosis. IRRI has identified the rice orthologue of a gene required for initiation of meiosis in yeast. Silencing of this rice gene may prevent entry in meiosis and allow AIformation.

Because Objectives 1a, 1b, and 2 were not attained completely, Objective 3 has not yet begun and will be postponed until Phase 3, which should focus on integrating discoveries in rice with those in Arabidopsis and natural apomicts such as Hieracium.