Rice Quality IV

by Rachelle Ward and John Oliver

NSW Department of Primary Industries

July 2008

RIRDC Publication No 08/132 RIRDC Project No PRJ-000482

© 2008 Rural Industries Research and Development Corporation. All rights reserved.

ISBN 1 74151 7214 ISSN 1440-6845

Rice Quality IV Publication No. 08/132 Project No. 000482

The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances.

While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication.

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This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165

Researcher Contact Details

Name: Rachelle Ward Address: NSW Department Of Primary Industries, PMB Yanco NSW 2703

Phone: 02 6951 2656 Fax: 02 6951 2719 Email: Rachelle.ward@dpi.nsw.gov.au

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.

RIRDC Contact Details

Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600

PO Box 4776 KINGSTON ACT 2604

Phone: 02 6271 4100 Fax: 02 6271 4199 Email: rirdc@rirdc.gov.au. Web: http://www.rirdc.gov.au

Published electronically in July 2008

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Foreword The Australian rice industry is dependent on the 4000 rice growers in the Murrumbidgee and Murray Irrigation Areas. In turn, the growers are dependent on the continual improvement of new varieties that excel in agronomic advantages and qualities for markets targeted by SunRice. The Quality Evaluation Program (QEP) is conducted as part of Rice Quality IV, and has contributed to the development of several cultivars that are in the final stages of release. Rice Quality IV has also ensured the QEP delivered contemporary knowledge on grain quality through adjunct research conducted to keep the program efficient and effective. Research in Rice Quality IV was conducted both locally and in collaborations with the International Rice Research Institute.

The milling component of the QEP was recognised as being labour intensive and required several discrete work stations. The Cervitec instrument had been newly developed and was recognised as having potential to implement efficiencies. The Cervitec is capable of using image analysis to predict physical properties of the grain and hence the measurement of several traits simultaneously rather than on several pieces of equipment, each requiring a separate operator. Implementation during this project has reduced the labour demands from 4.5 Full Time Equivalents (FTE) to 3.5 FTE during the mill season, and provided an additional grain cracking measurement.

The Australian rice industry prides itself on the quality of the rice produced, and it is essential to have the ability to characterise these qualities and to understand factors that influence these properties. Research in Rice Quality IV was conducted both locally and in collaborations with the International Rice Research Institute. Although the successive years of continual poor production has reduced the importance of being able to stockpile a quantity of rice for sale the following year, research into post-harvest studies was continued and a manuscript about the influence of post-harvest storage on milled grain quality will be submitted to a refereed journal. The article highlights the need to store milled rice below 20 °C for superior grain quality over time.

Other research was targeted towards screening several breeding lines for a range of starch synthesis genes including Waxy, Starch Synthase IIa (SSIIa) and Starch Branching Enzyme-1. Of particular interest is the potential of implementation of a screening tool for SSIIa to predict gelatinisation temperature which is important in determining cooking quality.

Rice Quality IV has helped foster a strong collaboration between Cereal Chemistry sections of International Rice Research Institute (IRRI) and NSW DPI. The Yanco Cereal Chemistry group is a member of the newly formed International Network for Quality Rice (INQR). This alliance has resulted in the Yanco Cereal Chemistry group being actively involved in several INQR taskforces, as well as in several small studies on the influence of nitrogen application on grain quality.

This report, an addition to RIRDC’s diverse range of over 1 800 research publications, forms part of our Rice R&D program, which aims to improve the profitability and sustainability of the Australian rice industry.

Most of our publications are available for viewing, downloading or purchasing online through our website:

• downloads at www.rirdc.gov.au/fullreports/index.html

• purchases at www.rirdc.gov.au/eshop

Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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About the Author After graduating in 1998 with a Bachelor of Science (Hons) from The University of Sydney, Dr Rachelle Ward worked in the pharmaceutical industry for several years before joining NSW Agriculture as a Technical Officer with the Rice Cereal Chemistry laboratory between 2001 and 2003. During this period she initiated post graduate studies at The University of Sydney under the supervision of Dr Melissa Fitzgerald and Prof Bob Gilbert. In 2007, Rachelle was awarded a PhD on her thesis titled ‘Impact of temperature and carbon dioxide on rice grain quality’. Rachelle commenced duties as Rice Cereal Chemist with NSW DPI at Yanco in January 2008.

Mr John Oliver, leads the Cereal Genetics and Improvement Unit for NSW DPI to underpin the productivity and profitability of the 4,200 NSW grain growers who annually produce 11 million tonnes of cereal grain worth $3.5billion. The Unit consists of 12 research scientists, and 52 technical support staff involved in:

• the breeding, selection and evaluation of improved varieties of wheat, barley, oats, rice and durum,

• industry driven research in genetics, cereal chemistry and economics, and • management of the Australian Winter Cereals Collection including curation, acquisition and

quarantine of germplasm from world wide sources for Australia’s cereal research effort. Mr Oliver represents NSW DPI in the research management Barley Breeding Australia, the NSW Agricultural Genomics Centre, the Australian Durum Wheat Improvement Program and the E H Graham Centre.

Acknowledgments The work conducted in Rice Quality IV was primarily possible through the dedication, thought and technical expertise of Margrit Martin, Judy Dunn, Kim Philpot and Hannah Blackburn.

We also thank Dr Melissa Fitzgerald for continued support, David Smith for assistance with securing and maintaining the ISO 9001 accreditation, Dr Arun Aryan for overseeing the project for 18 months and Dr. Russell Reinke for overseeing the project for 18 months. We also thank Dr Peter Snell and the rice breeding team for their support in the past 3.5 years.

We would like to acknowledge that support from the Rice Research and Development Committee of RIRDC has been unwavering.

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Contents Foreword ............................................................................................................................................... iii

About the Author.................................................................................................................................. iv

Acknowledgments................................................................................................................................. iv

Executive Summary.............................................................................................................................. vi

Introduction ........................................................................................................................................... 1 1. Quality Evaluation Program................................................................................................... 1

Cervitec ...................................................................................................................................... 1 Other Mill Room Equipment...................................................................................................... 4 Improvements in measuring cooking quality attributes ............................................................. 4 Parental Line Database ............................................................................................................... 5

2. QEP for concurrent RIRDC projects...................................................................................... 5 Nitrogen Trial (RIRDC project – DAN 207A)........................................................................... 5 Cold Tolerance Project (RIRDC project – DAN 1922-1) .......................................................... 5 Precision Agriculture (DAN 248A)............................................................................................ 6

3. Collaboration with IRRI......................................................................................................... 6 International Network for Quality Rice...................................................................................... 6 Nitrogen Study ........................................................................................................................... 8 Retrogradation Study................................................................................................................ 10 Gelatinisation Temperature Study............................................................................................ 10

4. ISO 9001 accreditation......................................................................................................... 12 5. Rice Storage ......................................................................................................................... 13 6. Genetics of Grain Quality .................................................................................................... 14

Starch Synthase IIa (SSIIa): ..................................................................................................... 14 Starch Branching Enzyme-1 (SBE-1) ...................................................................................... 14 Waxy ........................................................................................................................................ 14 Introduction of molecular screening in the QEP ...................................................................... 15

7. Fragrance analysis................................................................................................................ 16 8. References............................................................................................................................ 18

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Executive Summary What the report is about

The Australian rice industry is dependent on the 4 000 rice growers in the Murrumbidgee and Murray Irrigation Areas. In turn, the growers are dependent on the continual improvement of new varieties that excel in agronomic advantages and qualities for markets targeted by SunRice. The Quality Evaluation Program is conducted as part of Rice Quality IV, and has contributed to the development of several cultivars that are in the final stages of release. To ensure Quality Evaluation Program delivers contemporary knowledge on grain quality, it is vital that adjunct research is conducted to keep the program efficient is necessary. Research in Rice Quality IV was conducted both locally and in collaborations with the International Rice Research Institute.

The Final Report for Rice Quality IV highlights the achievements made throughout the lifetime of the project. The knowledge and advances made during the project have been well documented, and this report summarises these findings. Over 17 000 tests were performed on breeding lines as part of the Quality Evaluation Program, and through the introduction of new equipment the QEP has been significantly streamlined (Chapter 1). Beyond the QEP for the breeding lines for the Rice Improvement Program, the Cereal Chemistry team analysed a range of trials for other RIRDC projects as presented in Chapter 2. Chapter 3 reports on the participation in the newly formed International Network for Quality Rice which has expanded the scope of the Australian rice industry through interaction with the broader rice community. In Chapter 4, the recent acquisition and maintenance of ISO 9001 accreditation is reported. This accreditation confirms that the data generated by Cereal Chemistry is from a calibrated laboratory with documented procedures. Chapter 5 reports on a study into the post-harvest storage of rice can alter the rice quality - to address this concern a paper on storage has recently been submitted to a refereed journal. In Chapter 6, the progress made in exploring molecular markers is summarised. Screening breeding lines for grain quality is slowly moving towards the use of molecular markers instead of traditional wet chemistry techniques. Chapter 7 highlights the distribution of 2-acetyl-1-pyrroline throughout the rice grain in an attempt to learn about fragrant rice.

Who is the report targeted at?

The report offers an opportunity for the wider rice community to learn about the work conducted on behalf of the rice growers. The audience for the Farmers Newsletter and scientific papers are mutually exclusive, so this report offers an opportunity to share the knowledge across a broader audience. Also documents for posterity the advancements made so that future rice quality teams do not duplicate the work done.

Background

The milling component of the QEP was recognised as being labour intensive and required several discrete work stations. The Cervitec instrument had been newly developed and was recognised as having potential to implement efficiencies. The Cervitec is capable of using image analysis to predict physical properties of the grain and hence the measurement of several traits simultaneously rather than on several pieces of equipment, each requiring a separate operator.

The Australian rice industry prides itself on the quality of the rice produced, and it essential to have the ability to characterise these qualities and to understand factors that influence these properties. In recent years of irregular rice production, there was the option to stockpile a quantity of rice for sale the following year may be beneficial. While this is not a viable option with several years of continual poor production, research into post-harvest studies is necessary for Australia to maintain the reputation as an industry focused on quality.

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Aims/objectives

The three major objectives of Rice Quality IV were as follows: − To continue to evaluate the quality parameters of rice breeding lines and to improve the

screening techniques for more precision and efficiency. − The second objective was to conduct research into physical and chemical factors affecting rice

quality, ie grain cracking, retrogradation, gloss of cooked rice, lipid–amylose complexes, and aroma.

− The third objective of this program was to foster a strong and active collaboration between the Rice Improvement Program at Yanco and the new Cereal Chemistry section at IRRI.

The outcomes of these objectives will result in a faster delivery of new varieties to rice growers with improved yield and quality that reflect the demands of the market place. The ability to streamline the QEP is possible through attention to both the physical and cooking quality assessment of the breeding lines. The purchase of the Cervitec will allow several physical assessments to be combined in the one measurement to create labour savings during the milling season. The research component of Rice Quality IV involved several pilot studies to improve the efficiency and accuracy of the QEP. The major study of the project was a storage trial that aimed to understand of temperature and time on post-harvest grain quality. Methods used

The Cereal Chemistry mill room and laboratory is equipped with the ideal suite of equipment required for the work conducted in Rice Quality IV. Moreover, the lab and lab practices are ISO 9001 accredited.

Results/key findings

The Quality Evaluation Program has become more efficient with the introduction of the Cervitec to replace the length and width image analysis and the chalk system. This has reduced the labour demands from 4.5 Full Time Equivalents (FTE) to 3.5 FTE during the mill season, and provided an additional grain cracking measurement.

A manuscript about the influence of post-harvest storage on milled grain quality will be submitted to a refereed journal. The article highlights the need to store milled rice below 20 °C for superior grain quality over time.

Some research was targeted towards screening several breeding lines for a range of starch synthesis genes including Waxy, Starch Synthase IIa (SSIIa) and Starch Branching Enzyme-1. Of particular interest is the potential of implementation of a screening tool for SSIIa to predict gelatinisation temperature which is important in determining cooking quality.

Under the banner of the newly formed International Network for Quality Rice (INQR), there is a strong collaboration between Cereal Chemistry sections of IRRI and NSW DPI. This alliance has resulted in the Cereal Chemistry group being actively involved in several INQR taskforces, as well as in several small studies on the influence of nitrogen application on grain quality.

Implications for relevant stakeholders for:

The Australian rice industry prides itself on the quality of the rice produced. The quality and timeliness of the release of new varieties are important for the Australian rice industry to maintain its competitive edge over other suppliers. As the efficiency and accuracy of the Quality Evaluation Program continually improves, the number of breeding lines that can be screened each year can increase with minimal extra cost to the industry. Some improvements to the Quality Evaluation Program are leading to the real possibility of making selections earlier in the breeding program, this means that resources then can be redirected to identifying more difficult traits. Together with more research to support the Quality Evaluation Program such as post-harvest storage, the net effect will be

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more refined quality assessment for minimal increase in cost. Collaborative work such as participation in the International Network for Quality Rice underpins the research component and ensures the Quality Evaluation Program is cutting edge. The Australian rice breeding program also benefits through the improvements in the Quality Evaluation Program and the grower essentially have access to improved varieties quicker or more improvement in same time frame. The benefits of the work conducted in Rice Quality IV directly feeds into the Australian rice industry with the financial return directly to benefit local communities.

Recommendations

The research and evaluation of breeding lines is targeted towards a broad rice community that is inclusive of growers, marketers, and food manufacturers.

Opportunities to further streamline the Quality Evaluation Program exist as the full potential of the Cervitec is realised and as we further develop the parental line database. Continuation of the developments in Rice Quality IV, it is recommended that the following goals are achieved in subsequent projects.

1. Recommend development of medium grain brown rice calibration for millout. The Cervitec has an application to detect cracks in brown grain and preliminary evidence suggests that the degree of cracking is correlated with millout. If this proves correct for a larger samples set, it may be possible to screen brown grain for cracks before milling, discard samples with a high percent of cracking and limit the number of samples that require milling.

2. Recommend collaboration with another RIRDC project to use Near Infrared Spectroscopy (NIRS) to predict cooking qualities of rice. This would reduce the need for extensive sample preparation, but would yield less accurate results.

3. Recommend development of a bank of molecular markers for the rapid release of new varieties to respond to ever-changing environmental and marketing pressures.

New knowledge about the influence of storage time and temperature on grain qualities and which varieties are more susceptible to storage conditions allows marketers, consumers and food manufacturers an opportunity to review their storage practices.

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Introduction Rice Quality IV was a 3.5 year project spanning from January 2005 to June 2008. The major objectives of Rice Quality IV were as follows:

- To continue to evaluate the quality parameters of rice breeding lines and to improve the screening techniques for more precision and efficiency.

- The second objective was to conduct research into physical and chemical factors affecting rice quality, ie grain cracking, retrogradation, gloss of cooked rice, lipid–amylose complexes, and aroma.

- The third objective of this program is to foster a strong and active collaboration between the Rice Improvement Program at Yanco and the new Cereal Chemistry section at IRRI.

Throughout this final report it will become evident that despite the succession of leadership changes and the added workload through the acquisition of ISO 9001 accreditation, the Cereal chemistry group has managed to achieve these objectives.

1. Quality Evaluation Program

The function of the Quality Evaluation Program is to screen breeding lines in the Rice Improvement Program (DAN 881-1) for a range of physical and cooking qualities. The number of samples analysed each year for the duration of Rice Quality IV are listed in Table 1. The numbers of samples tested in the 2005 and 2006 seasons are typical. In 2007, only 2 980 samples were analysed in the mill room. This reflects the down size in the district trials as a result of the drought.

Table 1: Number of breeding lines screened through the Quality Evaluation Program.

2005 season 2006 season 2007 season Milled and physical

assessment 4 587 4 903 2 980

CT and gelatinisation temperature 968 1 735 703

Viscosity and amylose content 358 314 305

Cervitec At the beginning of Rice Quality IV, a Cervitec was purchased from FOSS. The Cervitec uses image analysis and artificial neural network to evaluate physical properties of the grain. The development of applications for the Cervitec is a combined effort between FOSS and our team. The progress of the Cervitec has been highlighted in two IREC Farmers Newsletters – Issue Number 168, Summer 2005 Edition and Issue Number 171, Summer 2006 Edition.

Chalk application

Chalk is the opaque belly of the grain that attracts significant price penalties, and is a key parameter analysed at the F4 stage of the breeding program. Previously, chalk was determined by capturing an image of a single layer of rice illuminated with a light box (Figure 1. Using a computer program, the proportion of opaque rice was calculated. Chalk analysis required 1 FTE (full time equivalent) throughout the milling season. The main problems associated with this image analysis system were the influence of the room (eg. different external light sources including shadows) and the daily calibration of the computer program which is subjective.

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Figure 1: Image analysis for chalk analysis.

The development of the chalk application for the Cervitec involved the collection of 3 000 images of single grains and dividing them into different chalk buckets by eye. The chalk buckets were < 10 % chalk, 10 – 25 % chalk, 25 – 50 % chalk, 50 – 75 % chalk and more than 75 % chalk (Figure 2). The chalk application has proved to be less variable than the previous method, and has been adopted into the Quality Evaluation successfully for three milling seasons.

> 75 % < 10 % 10 - 25 % 25 - 50 % 50 - 75 %

Figure 2: Chalk buckets used for the development of the chalk application.

Length and width application

Length and width was previously determined by image analysis of the brown grain and the length and width was determined through the conversion of pixel to mm (Martin et al, 1997). This system proved to be very accurate. Length and width analysis required 1 FTE (full time equivalent) throughout the milling season.

Using the Cervitec, the length application is measured on white grain (because it is measured at the same moment as chalk content) was initially based on buckets similar to the chalk application. Now, the length application uses digital analysis to relate the number of pixels to the length of the grain. The width calculation has not to been proved to be as successful as the length although work will be continued to improve this measurement.

Each year, the length of brown grain is measured by the Cervitec and is verified by using the ‘old’ length image analysis system. Variance between the length and width between the two systems is accounted for by Dr Peter Snell in the data analysis at the end of the milling season. This knowledge also allows breeders to predict the brown grain length of breeding lines.

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Grain cracking application

In the lifetime of Grain Quality IV, the grain cracking application has been established for medium grains only. An application for the grain crack detection in long grains requires further work by FOSS. Data generated from this application was used in a preliminary study designed to use grain cracking to predict mill out. The data from this trial is presented in Figure 3. The extraneous data points were found to be Arborio style breeding lines, in which the chalky appearance of the grain prevents crack detection. In the breeding program, lines that have a mill out of less than 50 % are not pursued. That is, around one third of the lines analysed in this trial tested in Figure 3 will be rejected by the breeding program. The ability to predict mill out through brown grain cracking has the potential to reduce the number of samples that require milling. When the correlation is validated over several trials and growing seasons, there is a possibility to significantly reduce the cost of the Quality Evaluation Program in the future.

0

5

10

15

20

25

30

30 40 50 60 70Millout (%)

Brow

n G

rain

Cra

ck (%

)

Figure 3: Correlation between mill out and brown grain cracking (R2 = 0.6293).

Broken grains application

For the broken grain analysis, the aim was to remove the need for the indent machine which separates the whole and broken milled grain for the calculation of mill out. This application was trialled in the 2005 milling season. Unfortunately, the broken grains clogged the grain delivery and the grain output mechanism of the Cervitec. This caused delay, inaccurate results and a need to regularly clean the Cervitec. In the 2006 and 2007 season, the broken grain application was used to evaluate the efficiency of the indent machine but not used in the calculation of mill out. The broken grain application may be reviewed when we receive a new wheel for the Cervitec.

Cervitec summary

In summary, the ability of the Cervitec to combine the tasks of analysing chalk and length and width has been successful and has reduced the number of staff required for the milling season from 4.5 FTE to 3.5 FTE. Outside of the milling season, the time required to maintain the calibration of the Cervitec is substantial however there is potential for more refined applications and additional applications such as long grain brown cracking to predict millout is a valuable tool. Ownership of the Cervitec allows an opportunity to participate in the International Network for Quality Rice (INQR) discussed in Chapter 3.

In the RIRDC project that flows on from Rice Quality IV, it is hopeful that the full potential of the grain cracking application used on brown grains can be used to develop a millout calibration.

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Other Mill Room Equipment The old de-huller (Satake) was able to completely dehull the paddy rice with constant adjustment of the distance between wheels. In 2006, a new de-huller was purchased to reduce the need to adjust the setting.

Prior to the 2008 milling season, the equipment in the mill room was assessed for wear and tear, and for any Occupational Health and Safety issues. Improvements to the mill equipment include the following:

- Dust collection and noise levels were improved on the KICE (an instrument that cleans harvested material into paddy by aspiration) and de-huller. The volume of the dust reservoir of the KICE was increased and the KICE was moved to a carpeted and dedicated room which should reduce the level of noise for the operator.

- The mill was observed to be worn out and a potential source of error in the use of the mill was recognised. A new mill casing and components were purchased to replace the worn out mill. A small study was required to ensure the performance of the mill matched the old mill. The previous milling method required the Operator change of weights on the mill arm part way through the 60s mill time which may introduce error into the result if the time the weight was changed differed between samples. The new mill method achieves the same results as the previous method without the need to change the weight midway through the mill time.

- The indent machine was altered to be more Operator friendly. Guards were extended, noise was reduced by placing rubber between parts that generated excess noise. The indent machine was mounted directly to the bench to stop movement and the handle of the indent machine were altered to be more operator friendly. The rubber on the scoop of the indent was replaced to improve the separation of broken and unbroken grains to improve the accuracy of the Percent Mill Out.

Overall the alterations to the Mill Room Equipment will lead to improved safety for the Operator and improved accuracy of the results generated from the Mill Room.

Improvements in measuring cooking quality attributes In late 2007, a review was conducted to estimate the time dedicated to each task in the Quality Evaluation Program. It was noted that it required 1 FTE 66 working days to maintain the computers and to transfer data between computers. The two most time demanding tasks were data management for amylose content and gelatinisation temperature. The efficiency of data management has been improved as follows.

- Amylose content is determined by measuring an amylose-iodine complex by absorbance at 620nm. Amylose content is then determined in reference to a standard curve. In 2006, a new UV/visible spectrometer were purchased by the Department of Primary Industries. In 2008, the new spectrometer and the balance used to weigh out flour for this assay is now connected to the same computer to allow faster collation of data into the single spreadsheet.

- Data could not be exported from the computer used to control the Differential Scanning Calorimeter (DSC). This computer was recently networked to a second computer to allow to be processed directly into a spreadsheet rather than written by hand for data entry at a later stage.

Typically, the CT PCR products are separated on an acrylamide gel and scored against a reference. The disadvantages of this system are the time consumed in setting up gel, exposure to the acrylamide and the error involved in scoring the separated gel bands. In 2007, the CT repeats were run on the CEQ 8000 – a sequencer located at the Wagga Wagga NSW DPI. This eliminates the need to set up an acrylamide gel and avoids inaccuracies involved with scoring the gels for CT repeat.

Protein is typically characterised using a SDS-PAGE gel to separate extracted proteins. The limitation of SDS-PAGE is that it was difficult to identify the difference in protein composition between varieties treated with different growth conditions. To overcome this problem, a promising new tool,

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the ‘Bioanalyser 2100’, was trialled at Charles Sturt University in Wagga Wagga. Unfortunately, the Bioanalyser is still only set up to look at high molecular weight proteins. For small molecular weight proteins such as small heat shock proteins in wheat, the DNA chip on the Bioanalyser would be able to detect the proteins synthesised. Such work could be recommended for future work.

Parental Line Database There are around 200 parental lines that feature strongly in the Rice Improvement Program (DAN 881-1). Currently the progeny of crosses that have promise for a pre-determined quality are analysed as part of the QEP. Of these progeny, often varieties with similar quality attributes but different agronomic features are crossed. For example, Amaroo a variety with CT 19 crossed with Langi also a CT 19 variety may yield progeny that differ in plant height but the CT would still be 19. In this case, it would not be necessary for the progeny to be analysed for CT. The ability to be more discerning with samples analysed through the QEP will have the potential to reduce the salary and operation costs required for the QEP each year.

A database of for the germplasm from the seed store has long been established with quality data including amylose content and CT of each variety. In 2008, a smaller database with current parental lines has been created and will be continually added to as additional markers are introduced into the QEP. So far, a chunk of the Waxy gene that contains the CT repeat, single nucleotide polymorphism (SNP) at the splice site of intron 1, and a second SNP within the intron 1 has been sent to Southern Cross University for sequencing. The data will be integrated into the parental line database so that the molecular footprint of each parental line is expanded (see Section 6 for further details).

2. QEP for concurrent RIRDC projects

Typically the QEP is focused only on the screening of breeding lines that develop through the Rice Improvement Program. In addition, the Cereal Chemistry team supported and collaborated with other RIRDC projects through the analysis of grain using methods typically used in the QEP. The data generated for other RIRDC projects was provided to the respective RIRDC projects and is briefly described below.

Each program supported the milling of their samples thought the provision of staff at various stages of the milling season each year. For this help, we are grateful.

Nitrogen Trial (RIRDC project – DAN 207A) Grain from a nitrogen trial conducted by Dr Ranjith Subasinghe in 2005/06 was milled by the Cereal Chemistry laboratory. The millout, colour, chalk, length and width of the grain were determined for 233 samples. A summary of all nitrogen trials showed that the chalk content had the most variability. In particular, the proportion of grains that contained less than 25 % chalk varied the most which suggests that N fertilisation plays a role in the chalk content of the grain.

Cooking qualities were analysed for only the 0 N and the 270 N samples. Viscosity and hardness were determined for a selection of 85 samples. Gelatinisation temperature and amylose content were measured for 96 samples. A further 12 samples were treated with protease and viscosity was recorded. A glance of the data shows that nitrogen rates did little to alter amylose content and gelatinisation temperature but cooking quality parameters showed a known response.

Cold Tolerance Project (RIRDC project – DAN 1922-1) In 2007, 1800 breeding lines from the cold tolerant program were assessed for heterozygosity of the CT repeat located in the non-coding region of intron 1 (Ayres et al, 1997). Knowledge of the heterogeneity of the lines allows breeders to gauge if the lines have segregated into discrete cultivars. Heterozygosity was determined by sequencing the PCR product containing the CT repeat. As with a gel, the sequencer (CEQ located at NSW DPI in Wagga Wagga) separates the CT repeats and records the signal at different elution volumes with a CT repeat of 8 eluting before a CT repeat of 20. A homogenous product will feature as a single peak, and a heterogenous symbol as a double peak as featured in Figure 4.

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(a) (b) Figure 4: Raw data from the CEQ sequencer showing a variety with heterogeneity (a) and homogeneity (b). Precision Agriculture (DAN 248A) In 2007, grain was milled for a trial conducted as part of the RIRDC project Precision Agriculture (DAN 248A). The nitrogen trial was conducted across four farms (Draper, Houghton, YAI and McCaughey) and had two nitrogen rates. Of interest is the chalk data from each farm and N application rate presented in Table 2. Table 2: Chalk data for RIRDC project DAN 248A.

Farm Nitrogen Rate

Total Chalk

0-10 % chalk 10-25 % chalk > 25 %

chalk Comments

YAI 0 7.2 ± 1.7 63.4 ± 7.5 33.6 ± 6.3 3.0 ± 1.9 Good Translucency YAI 120 5.4 ± 1.6 76.8 ± 6.6 18.7 ± 5.6 4.5 ± 1.9 Good Translucency

Draper 0 1.3 ± 0.6 95.0 ± 1.9 3.7 ± 1.3 1.3 ± 0.8 Draper 120 6.7 ± 1.4 75.7 ± 4.5 16.9 ± 3.8 7.5 ± 2.3 Grain Not Translucent

McCaughey 0 13.6 ± 5.0 32.9 ± 22.0 61.4 ± 19.1 5.8 ± 3.7 McCaughey 120 11.8 ± 2.3 44.9 ± 9.9 48.4 ± 8.3 6.6 ± 2.0 Houghton 120 3.3 ± 1.2 86.8 ± 4.5 10.4 ± 3.3 2.8 ± 1.5

At the higher nitrogen rate at YAI and McCaughey total chalk decreased as a greater proportion of grains had 0-10 % chalk and a decrease in grains with 10-25 % chalk. The proportion of grains with more than 25 % chalk has not changed. In contrast, grain from the high nitrogen trial of the Draper farm recorded an increase total chalk due to a decrease in the proportion of grains had 0-10 % chalk and a increase in grains with 10-25 % chalk. It was observed during the analysis of the grain that the grain from the high nitrogen trial on the Draper farm was not translucent and questioned if the moisture content of the grain was not 15 %. A control trial at the Houghton was not available for analysis. A study to understand the relationship between high nitrogen content and lower chalk may uncover a pathway to understand the processes that cause chalky grains, and thus offer a solution to tackle this age-old problem.

3. Collaboration with IRRI

International Network for Quality Rice The third objective of Rice Quality IV was to foster a relationship between Rice Improvement Program at Yanco and the cereal Chemistry section at IRRI. As part of this collaboration, Margrit Martin visited IRRI to use novel instrumentation and to compare protocols for rheological studies. A travel report has been submitted in which the key benefits identified were: • Samples of rices grown at Yanco under varying conditions of nutrition were analysed on equipment not available in Yanco.

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• A new source for capsules for the Dental amalgamator was identified (unavailable in Australia for past few years) • Observed a new small mill using a paint shaker which could be of great benefit here to mill large numbers of small samples simultaneously. • Accessed a small sample of 2-AP (2Acetyl-1-pyrroline), synthesized in Japan, which will be invaluable for quantifying the major fragrant compound in rice on the GC. This compound is very expensive in Australia • Identified that the analysis for amino acids analysis could be implemented in Yanco with the existing HPLC system and detectors. This relationship was further fostered under the umbrella of the International Network for Quality Rice (INQR). The INQR is an initiative outside the traditional American Association of Cereal Chemists aimed towards the development of international standards for evaluation of rice quality. There are several taskforces within the INQR of which Rice Quality IV has been involved in several. Physical Taskforce

The Physical Taskforce is tailored to the development of an international application for the measurement of physical qualities of the brown and milled grain using the Cervitec. The Cereal Chemistry group at Yanco has been instrumental in the supply of thousands of images that were used to develop existing applications and for use in the development of further applications useful to the Australian rice germplasm.

Amylose Taskforce

The Amylose Taskforce is aimed towards the agreement of a universal standard to quantify amylose content. Cereal Chemistry group at Yanco was involved in Round 1 of the project which involved around 28 rice quality labs around the world to use there standard method to measure the amylose content of 17 samples. The variability in results encouraged Round 2 in which samples were sent with a defined procedure. Unfortunately we were unable to participate in Round 3 because the Size Exclusion Chromatograph required for the analysis is currently uncommissioned.

Sensory Taskforce

The Sensory Taskforce offers an opportunity to have Australian rice judged by a panel of trained sensory analysts. Recently, two long grain Australian varieties that vary in CT and theoretically cooking class were submitted to the trial. It is expected that the sensory trial will offer an understanding about the subtle differences between Pelde (CT 18) and Langi (CT 19). Data from the sensory evaluation is not expected within the timeframe of this proposal.

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Nitrogen Study A collaborative work with Margrit Martin, Russell Reinke and Melissa Fitzgerald culminated in a poster presented at the 2006 Australian Cereal Chemistry Conference. The poster is summarised below.

Title:

Balance between protein and starch composition in rice grains

Introduction:

Two alleles are described at the waxy locus (Sano 1984) – Wxa which maintains amylose content under different temperatures of grain-filling, and Wxb which does not (Larkin and Park 1999). Doongara is the only Australian variety that carries the Wxa allele, and we have observed significant variation in amylose content over many seasons (Figure 1). High temperature and N nutrition are the two factors that contribute the most to rice growth. N nutrition can be managed and its effects on plant growth, harvest index and protein content are known. The effect of N-nutrition on increasing the protein content of rice directly affects several of the cooking properties of the rice (Martin and Fitzgerald 2002). Figure 1 also shows the variability of amylose in a variety carrying the Wxb allele – Amaroo. The variability in amylose content of Amaroo can be explained by temperature differences between seasons. Given that Doongara carries the Wxa, such variability is unexpected. In this study we used a collection of Wxa carriers, and 2 Wxb carriers to determine if the rate of N nutrition affects the proportion of amylose in the rice grain.

0

2

4

6

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10

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15 16 17 18 19 20 21 22 23 24 25 26 27

% Amylose

Freq

uenc

y

Amaroo Doongara

Figure 1: Distribution of amylose content (%) in Amaroo (Wxb) and Doongara (Wxa) from different trials over 5 years. (experimental and environmental error combined)

Materials and Methods:

Pots (20cm diameter) were filled with a mixture of soil (clay) and sand (washed river sand) to give a minimum initial nutrient content. The pots for the low nitrogen (LN) treatment were flooded and drained to lower nutrient content further. Four pots were sown of each of the nine varieties for the LN treatment and four pots grown with high N (HN). Seedlings were thinned to 5 per pot and then plants were grown in controlled temperature environment until maturity. The temperature by day was 30 °C by night was 20 °C. Grains were harvested at maturity, dehulled, milled and ground. Nitrogen was measured with the Dumas combustion method and protein was calculated. Amylose was measured by iodine binding.

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Results and Discussion:

Table 1: Nine Varieties from different Amylose classes

Variety CT Waxy allele Amaroo 19 b Opus 17 b Doongara, TJ9 14 a IGT, Rexmont, IR38, IR28, IR36 11 a

Table 1 shows clearly a significant increase in the protein content of grains of all varieties when grown with high N nutrition. In all varieties of intermediate and high amylose, high N nutrition caused a decrease in amylose content (Table 3, Figure 2). In the two varieties carrying the Wxb allele, the loss of amylose was small (Table 3, Figure 2); presumably a loss in amylopectin compensated for the increase in protein. Both N treatments produced healthy grain that would meet the specifications of receival sites, however the differences in the composition of the grain would lead to noticeably different cooking properties. The data indicate that protein synthesis is a stronger sink for carbon than starch synthesis, and given the difference in varieties, there could be genetic variation for the strength of the protein sink. Varieties with higher amylose, and thus lower amylopectin, sacrifice amylose to meet the demands of protein synthesis. Different environmental conditions and different management conditions can influence the availability of N in flooded soils, so we expect seasonal variation in the N content of rice plants and rice grain. However, the data indicate that differences in protein content do not decrease amylose and amylopectin proportionately, but for intermediate and high amylose rices, higher protein comes at the expense of amylose. For low amylose varieties, presumably amylopectin is expended. Thus expression of the Wxa allele, while not affected by high temperature, is susceptible to nutritional conditions, explaining the seasonal variability we observe in the amylose content of Doongara.

TJ9 AmarooDoongaraRexmont IR28 IR38 Opus

IGT IR36

TJ9

Amaroo

DoongaraRexmont

IR28

IR38 OpusIGT

IR36-6

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% Protein increase % Amylose decrease

Figure 2: Increased Protein content causes decrease in Amylose content

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Table 3: Effect of N nutrition on amylose and protein content in all 9 varieties.

Protein Content (%) Amylose Content (%) Variety Low N High N Low N High N

Opus 7.26 13.51 18.66 16.57 Amaroo 6.96 11.19 19.56 19.23 IR 28 7.85 13.33 23.73 19.05 Rexmont 9.64 14.88 25.5 22.37 Doongara 8.51 13.33 26.25 23.37 TJ9 6.96 10.89 26.7 24.11 IR 36 8.57 17.73 29.69 23.94 IR 38 9.82 15.77 28.69 26.15 I-Geo-Tze 7.26 15.83 29.97 26.75 Retrogradation Study In addition to collaborative work under the auspices of the INQR, a project conducted and reported as Chapter 6 of Rice Quality 3 was further developed and has since been published in a refereed journal.

Philpot, K., et al., Environmental factors that affect the ability of amylose to contribute to retrogradation in gels made from rice flour. J. Agric. Food Chem., 2006. 54 (14): p. 5182-5190.

Gelatinisation Temperature Study A collaborative work with Margrit Martin, Melissa Fitzgerald and Russell Reinke culminated in a poster presented at the 2007 Australian Cereal Chemistry Conference. The poster is summarised below.

Title:

Does sample preparation affect gelatinisation temperature?

Introduction:

Gelatinisation Temperature (GT) is an important factor controlling cooking quality in rice. It contributes to cooking time, energy required for cooking and texture of cooked rice. It is one of the primary traits of cooking quality that breeding programs use for selection. Successful selection depends on accurately measuring GT. We determine GT of 1 500 rice breeding lines with a Differential Scanning Calorimeter (DSC) every season. Sample size often dictates the grinding method used. The following work will demonstrate how particle size and/or starch damage through different grinding affects accuracy and precision.

Materials and Methods:

Three commercial rice varieties, Doongara, Amaroo and Langi were ground using five different methods: 5mg of above flour samples with 10 µl of water in five replicates were sealed in a 40 µl aluminium pan and heated from 50-100 °C in a Mettler DSC while the endotherm was recorded.

Table 1: Five methods to produce flour with different particle size.

CapMix 3-30 grains minimal <200 µm

Mill Sample size Sample loss Particle size

Rocklab 1- 1000 grains none <100 µm

Cyclotec 1093 2g - unlimited extensive <500 µm (screen)

Wiley mill 0.2g - unlimited minimal <500 µm (screen)

Razor Blade

½ grain none Variable slice

Microscopic Images

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Results and Discussion:

Surprisingly, on every sample with the finest particle size, it was impossible to detect an endotherm, thus impossible to determine the peak temperature – gelatinisation temperature. With the largest particle size – a slice of grain, it was possible to detect a large endotherm but the reproducibility was poor. The samples with the intermediate particle size, specially > 200 µm, gave a good endotherm with a clear and reproducible peak temperature.

Figure 1 shows that gelatinisation temperature is affected by particle size or grinding method.

It is unclear why the sample with the finest texture shows no endotherm. Is the crystalline structure lost under heat and pressure of the friction/impact mill?

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atin

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Amaroo Langi Doongara

Rocklab Capmix Cyclotec Wiley Razor

The slight difference between the Cyclotec mill and the Wiley mill can be attributed to particle size disposition or shape through the difference in the grinding principle. The Cyclotec mill forces the grain against an abrasive ring where the Wiley mill cuts the particles between stationary and rotating blades.

The poor reproducibility in the last sample preparation is understandable as we measure only one segment of one grain. The crystallinity of amylopectin is expected to differ slightly between different parts of the grain or grains ripening at different times.

Conclusions:

In quality evaluation programs it is crucial that (i) sample preparation is standardised and (ii) that knowing the particle size and testing this regularly becomes a component of the quality management system.

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4. ISO 9001 accreditation

An initiative of NSW Department of Primary Industries is for laboratories to gain ISO 9001 accreditation which satisfies elements of Occupational Health and Safety and Quality Management Systems. The Cereal Chemistry group were pro-active in gaining ISO 9001 certification. Key achievements for the group were:

• Laboratory equipment calibration program implemented during 2005.

• Chemicals store content was reduced to a manageable level. All chemicals are labelled and stored according to their safety class.

• Quality Manual complying with ISO 9001 completed 26 July 2006.

• 27 Standard Operating Procedures completed prior to September 2006.

• NCSI external audit of Rice Cereal Chemistry Quality Management System on 22 September 2006.

• NCSI recommended ISO 9001 Certification.

• ISO 9001 Certificate issued to Rice Chemistry on 1 May 2007.

• Quality Manual reviewed and revised April 2008.

• 23 Standard Operating Procedures reviewed and revised May 2008.

• 17 Risk assessments completed May 2008.

The completion and regular review of Standard Operating Procedures for the major tasks conducted by the Cereal Chemistry groups limits the loss of IP from changes in staff. The SOPs are written by the technician that performs the process and contains handy site-specific information about the task as well as useful tips that will assist in the successful and efficient performance of the task. The Risk Assessments for both the tasks and any chemicals used to perform the task will minimise the associated OHS risks to the task. The data generated from a certified ISO 9001 lab provides confidence and satisfaction for the recipient, and has become an essential requirement for the application of external funding. So, while the application process for ISO 9001 is exhaustive and requires regular review to maintain the Certification it is beneficial for the Cereal Chemistry to be accredited.

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5. Rice Storage

Rice is bred and grown for a specific product, and knowledge of the changes in grain quality during post harvest handling is essential for successful marketing of that product. During Rice Quality IV, an experiment was conducted on the effect of storage temperature and time on cooking qualities. Unlike many storage trials, in this study seven milled Australian rice varieties that differed in amylose content, gelatinisation temperature and grain dimension were used with the aim of isolating the characteristic, or perhaps combination of characteristics that are less prone to undesirable post-harvest changes to grain quality. As this work is currently being developed into a manuscript for publication in a referred journal, a brief summary of the major outcomes of the work is reported here.

Milled grain was stored at 4 °C, 20 °C and 37 °C for 21 months. Every three months a sub-sample of the milled grain was ground and the pasting characteristics, hardness of the retrograded gel and gelatinisation temperature were analysed. It was found that most quality characteristics were similar for the 4 °C and 20 °C storage temperatures, but differed for grain stored at 37 °C. For milled rice stored at 4 °C and 20 °C, pasting temperature remained unchanged, peak and final viscosity increased at the same rate so setback remained unchanged. Gelatinisation temperature and pasting temperature were maintained as did the hardness of the retrograded gel.

Milled rice stored at 37 °C showed an increase in pasting temperature, setback and final viscosity and a decrease in peak viscosity. In comparison to medium grain, low gelatinisation temperature varieties, the long grain varieties that have a high gelatinisation temperature exhibited a different pattern of change for most characteristics. The soft-cooking milled rice retrograded less than the hard-cooking rice. In the manuscript it will be discussed that grain dimension, amylose content and gelatinisation temperature all play certain role in explaining differences between functional properties in varieties stored at various temperatures, so it is important to consider the quality as a sum of changes to the flour as a whole rather than only one component of the flour.

This study also highlighted that basic assumptions about pasting properties become invalid for milled grain stored at 37 °C. For example, amylose content does not correlate with peak viscosity or setback. For hard-cooking rice stored at 37 °C, the setback increases the least and for soft-cooking rice stored at 37 °C, the setback increases the most. Thus it is important to re-evaluate the interpretation of results from rice stored at higher temperatures.

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6. Genetics of Grain Quality

Gene-specific markers are a useful introduction into the selection process for the Breeding Program. A proven example of gene-specific markers is the CT repeat. Since 2001, the CT repeat has been determined for upwards of 1 000 breeding lines each year to ensure the segregation of new cultivars, to trace variability between cultivars and to predict amylose content and cooking class. The advantages of using markers in a breeding program are:

- Little sample is required and thus allow the prediction of grain qualities early in the breeding program. For example, amylose content and viscosity are measured at the F6 generation but could be predicted with markers as early as the F4 generation.

- The time and expense of molecular screening resides in the extraction of suitable DNA, so the introduction of additional markers is cost-effective in terms of dividing the cost of the DNA extraction over several markers and the savings made by reducing the number of samples that require wet chemistry (eg. each measure of gelatinisation temperature costs around $2.20).

- In the course of Rice Quality IV there have been two occasions where molecular markers have been used to characterise otherwise similar rice varieties. The first was to identify Quest 19 and the other was to identify if seed provided to growers was Amaroo or Jarrah.

Several small scale studies using molecular markers have been conducted in Rice Quality IV. These studies are summarised here.

Starch Synthase IIa (SSIIa): Currently, approximately 800 breeding lines are measured by Differential Scanning Calorimetry (DSC) at a labour cost of ~$5500 and operating cost of ~$4000. Gelatinisation temperature is the heat required to cook the grain, and is related to the structure of A-chains in amylopectin synthesised by Starch Synthase IIa (Umemoto et al., 2004). In a collaborative work seventy rice cultivars were phenotyped for gelatinisation temperature by the Cereal Chemistry group (Yanco, Judy Dunn) and the corresponding DNA was genotyped by Southern Cross University (USC-6A). Results showed that varieties with a high gelatinisation temperature (~78 °C) correspond to a Single Nucleotide Polymorphism (SNPs) combination of G/G/GC and A/G/GC and a low gelatinisation temperature (~70 °C) corresponds to A/G/TT and A/A/GC.

This work has been published in a referred article (Waters et al., 2006) and highlighted in an IREC Farmers Newsletters Issue 174, 2006-07 Summer Edition.

Starch Branching Enzyme-1 (SBE-1) Starch Branching Enzyme (SBE-1) is used in the synthesis of amylopectin chains. There are two known alleles of SBE-1, the promoter region of SBE-1 contains a transposon (Tourist Os6) that is present in japonica varieties but absent in indica varieties. Work conducted by two visiting students, Annabel Boret and Gayle Warnock, under the supervision of Dr. Aryan screened the distribution of these SBE-1 alleles in part of the Australian germplasm.

In the IREC Farmers Newsletters Issue 174, 2006-07 Summer Edition, it was reported that Amaroo, Bogan, Pelde and Illabong amongst others contain the Tourist Os6 transposon common to japonica varieties and the SBE-1 of Doongara and several parental lines do not contain the transposon to indicate the variety is indica. As reported in the Issue 177, 2007-08 Summer Edition of the IREC Newsletter and was presented as a poster at the Cereals conference in 2006 (Warnock et al., 2006), no correlation between SBE-1 and starch synthesis or any cooking quality has been established.

Waxy Amylose content has long been mapped to the splice site of intron 1 (Isshiki et al., 1998; Hirano et al., 1998). A low amylose rice has a T at the splice site and is classified as a Wxb allele, while intermediate and high amylose rice carry a G at the splice site known as a Wxa allele. Varieties with a low amylose content exhibit a negative viscosity setback and those with a high amylose content show a positive

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setback (Juliano at al., 1964). From this literature, it is expected that a low amylose rice that carries the Wxb allele will have a negative setback just as a high amylose rice that carries the Wxa allele will have a positive setback. This work was reported in the IREC Newsletter Issue 177, 2007-08 Summer Edition.

Introduction of molecular screening in the QEP The advancement of the Australian rice breeding program is dependent on the introduction of a well developed, robust, and diverse molecular screening program. To date, this work has not been introduced into the routine molecular screening component of the Quality Evaluation Program. There are several reasons for this:

- The starch characteristic and this cooking quality phenotype that is mapped to the SBE-1 alleles remains unknown so its value in the breeding program is questionable.

- The sodium hydroxide extraction technique for CT yields poor quality DNA unsuitable for more sophisticated SNP analysis. So, before routine testing, a high throughout, high quality DNA extraction technique suitable for use in all marker screens needs to be evaluated. In 2008, experiments were initiated to investigate four DNA extraction techniques for cost and suitability to the primers expected to be used in the QEP.

- The SSIIa protocol developed by Southern Cross University is currently being optimised by Cereal Chemistry (Kim Philpot). Introduction into the Quality Evaluation program is limited to future funding.

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7. Fragrance analysis

The third objective of Rice Quality IV was to establish a protocol for measuring aromatic compounds in rice. This work was presented in poster format by Kim Philpot, Arun Aryan and John Oliver to the RACI Cereal Chemistry Conference in 2005.

Title:

Grain quality of fragrant rice: Distribution of 2-acetyl-1-pyrroline in tissues of paddy rice

Introduction:

2-acetyl-1-pyrroline (2AP) is thought to be the major aromatic compound contributing to the ‘nutty–popcorn’ flavour in fragrant rices. Although the amount of 2AP present in a fragrant rice grain can be influenced by the growing season and storage conditions, it is mainly dependant on varietal genetics. For breeding of fragrant rice varieties 2AP is highly sought after in potential crossbreds, and is identified by extracting rice flour with dichloromethane (DCM) then analysed by gas chromatography. The entire process requires the rice sample to be harvested, stored, kiced, de-hulled, milled and ground prior to 2AP extraction. This can mean it is many weeks before rice breeders have any information on crossbreds, and often requires the consumption of large quantities of limited seed.

The aim of this project was to estimate the amount of 2AP in different parts of rice paddy to identify a suitable tissue for rapid screening of aromatic rices, and to obtain comparative results for a follow up study aiming at extracting 2AP from green tissues in the field.

Materials and Methods:

Australian cultivars Kyeema (fragrant) and Langi (non-fragrant) were used in this study. Paddy rice was separated into hull, brown grain, white grain, bran, brown flour and white flour (Figure 1). For extraction of aromatic compounds, a solvent stock of dichloromethane (DCM) containing 1ug/ml of Collidine (2, 4, 6-trimethylpyridine (TMP)) was used. Approximately 300mg of tissue samples in 750ul of the DCM stock solution were incubated at 85°C for 2.5 hours. Extracted samples were then analysed on an Agilent 6890 GC fitted an HP-5 wide bore column. Helium was the carrier gas and 2ul of sample was injected on the column.

Results and Discussion:

The amount of 2AP present in the various parts of paddy rice was estimated in relation to the known concentration of TMP (1ug/ml) using the ratio: peak area of 2AP /peak area of TMP. Relative amounts of 2AP in the extracts from different parts of Kyeema paddy are shown in Figure 2. Although brown flour showed the highest amount of 2AP, a significant amount was also present in the hulls that could be used for screening fragrant crossbreds. In contrast to Kyeema, different tissues from Langi did not show the unique peak for 2AP (data not shown). The extracted amounts of 2AP from flour samples was much higher than from the whole grain and bran. This is probably related to greater surface area of flour sample and lower permeability of DCM through the bran tissue.

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Figure 2: Relative amount of DCM-extracted 2AP from different parts of Kyeema paddy

The most important result here is the hulls of fragrant line (Kyeema) contain a significant amount of extractable 2AP as compared to that in Langi (Figure 3). Thus, hull tissue could be used as a suitable screening material for the presence of 2AP.

Figure 3: Relative amount of 2AP in Langi and Kyeema hulls

Conclusions:

In the fragrant rice, 2AP is present in all the tissues of paddy rice, while in the non-fragrant line it was undetectable. In fragrant rice, a significant amount of 2AP is present in the hull tissue, which could be utilised in screening fragrant crossbreds thus saving limited grain samples. Bran tissue contains high levels of lipids that appear to interfere with DCM-extraction of 2AP. Therefore DCM may not be an ideal solvent for the extraction of 2AP from bran or whole brown grain. This preliminary study has shown that the level of 2AP in the hull is a significant indicator of the presence or absence of 2AP in crossbred rice lines.

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8. References

Ayres, N.M., et al., Microsatellites and a single-nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germ plasm. Theoretical & Applied Genetics, 1997. 94 (6-7): p. 773-781. Hirano, et al., A single base change altered the regulation of the waxy gene at the posttranscriptional level during the domestication of rice. Molecular Biology & Evolution, 1998. 15(8): p. 978-987. Isshiki, M., et al., A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron. Plant Journal, 1998. 15(1): p. 133-138. Juliano, B.O., et al., Some physicochemical properties of rice in South East Asia. Cereal Chemistry, 1964: p. 275-285. Larkin, P D and Park, W D. Transcript accumulation and utilization of alternate and non-consensus splice sites in rice granule-bound starch synthase are temperature-sensitive and controlled by a single-nucleotide polymorphism. Plant Molecular Biology, 1999. 40, (4): p. 719-727. Martin, M., et al. Image Analysis to assess dimensions of rice grains. in 47th Australian Cereal Chemistry Conference. 1997. Perth: Cereal Chemistry Division. Martin, M and Fitzgerald, M A. Proteins in rice grains influence cooking properties! Journal of Cereal Science, 2002. 36: p. 285-294 Sano, Y. Differential regulation of waxy gene expression in rice endosperm. Theoretical and Applied Genetics, 1984. 68, (5): p. 467-473. Umemoto, T., et al., Natural variation in rice starch synthase IIa affects enzyme and starch properties. Functional Plant Biology, 2004. 31: p. 671-684. Warnock, G.F., et al. Distribution of starch branching enzyme (Sbe-1) allelles in Australian rice cultivars. in 56th Australian Cereal Chemistry Conference. 2006. Freemantle: Cereal Chemistry Division, Royal Australian Chemical Institute. Waters, D.L.E., et al., Gelatinization temperature or rice explained by polymorphisms in starch synthase. Plant Biotechnology Journal, 2006. 4(1): p. 115 - 122.

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