ENHANCEMENT OF RICE (ORYZA SATIVA) NUTRITIONAL VALUE AND GRAIN QUALITY AND DEVELOPMENT OF HERBICIDE-RESISTANT RICE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016131
• Project Start Date: May 23, 2018
• Project End Date: Apr 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100

Performing Department
Rice Research Station

Non Technical Summary
A high-protein rice cultivar, Frontière, Reg. No. CV-150, PI 674794, was successfully developed by this project and was released in 2015. This is a non-GMO rice and the first high-protein rice cultivar in the world developed for commercial production. Plant Varietal Protection for this cultivar has been obtained from the USDA office (Certificate No. 201500310).The patent application also has been issued by the United States Patent and Trademark Office(USPTO No. 9888637). Frontière was developed from the cultivar Cypress through induced mutation. It has an average protein content of 11% which is about a 53% improvement above the protein content of typical U.S. cultivars, such as Cocodrie, Cheniere, Catahoula, Francis, and Cypress.Promising advanced lines with higher protein and improved yield are currently being evaluated in multiple locations for potential releases. These new high-protein lines were developed in different genetic backgrounds and have specific grain quality profiles to meet specifications of the food industry. The success of developing the first high-protein rice cultivar has provided the base for fine-tuning grain quality characteristics to improve marketing high-protein rice.Provisia (ACCase inhibitor) riceis resistant to anew herbicide technology that will be introduced soon. With thistechnology farmers can rotate with the Clearfield Production System. It can provide flexibility to plant rice on more acres while rotating different herbicide modes of action [Acetolactate Synthase (ALS) and Acetyl CoA Carboxylase (ACCase) Inhibitors]. Our research will focus on developing herbicide-resistant lines that are different from both Clearfield and Provisia technologies, i.e. resistant to glyphosate (Roundup) and S-metolachlor (Dual Magnum). The availability of new herbicide-resistant rice will further improve the flexibility of managing weeds and will lower rice production costs. It also will slow resistance evolution in weedy rice. New herbicide-resistant rice will allow for a longer rotational period for each herbicide to which will reduce potential carryover from a particular herbicide.

Animal Health Component

45%

Research Effort Categories

Basic

20%

Applied

45%

Developmental

35%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1530	1081	75%
202	1530	1080	25%

Knowledge Area
202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1530 - Rice;

Field Of Science
1081 - Breeding; 1080 - Genetics;

Keywords

rice

high protein

herbicide resistance

amino acids

Goals / Objectives
The major goals of this project are:Develop, evaluate, and release new high-protein rice cultivar(s) thatimprove health benefitsand grain qualityto fulfill specific product requirements.Develop and characterize new rice lines that are resistant toselect classes of herbicides, (ieglyphosate and S-metolachlor), to provide a wider choice of weed control and to reduce costs.

Project Methods
Development of Cultivars with Improved Nutritional Value1.Mutational Breeding: The high-protein rice lines will be developed from cellular selections by exposing them to elevated toxic levels of S-2-aminoethyl-L-cysteine (AEC), a sulfur analog of L-lysine from 0.5 to 1.5 mM. Cells surviving from the treatment will be rescued and plated onto semi-solid regeneration media to produce plantlets.2. Conventional Breeding (Selections, Line Purifications, and Advancement): In each generation following mutation and cellular selection (M1 to M6), the high-protein rice line will be selected for consistent high grain protein content, uniformity, and purity of its agronomic traits. Selected panicles will be subjected to protein analyses for selection and purification. The seed from selected panicles of M2 generation and above will be bulked and then used in replicated yield trials. The promising lines will enter Preliminary Yield (PY) trials at the HRCRRS, and Multi-Location Yield (MLY trials).3. Measurement of Nutritional Grain Quality: Amino acid profiling: Amino acid profiling will be conducted using samples collected from yield tests according to the method of Zhu and Galili. Some samples will also be sent to a commercial laboratory for confirmation. Beckman 6300 Amino Acid Analyzers with post-column, ninhydrin derivatization will be used to analyze for 16 amino acids, hydroxyproline, hydroxylysine, cyst(e)ine as cysteic acid, and tryptophan.4. Isolation of the different fractions of seed storage proteins: Major rice protein fractions, albumin, globulin, prolamin, and glutelin will be extracted from seed powder and precipitated proteins will be centrifuged at 12,000 g for 15 min, dried briefly in a speed vacuum, and resuspended in SDS sample buffer. For each protein fraction, 10 µL from each line will be subjected to SDS-PAGE according to the manufacturer's instructions.5. Analyze the content of dietary fibers, vitamins, minerals, specific oils (γ-oryzanol), and disease fighting phyto-nutrients among high-protein lines: Total dietary fiber will be determined by the Association of Official Analytical Chemists (AOAC) enzymatic gravimetric method (AOAC Method 991.43, 1992).Development of Herbicide-Resistant Lines1. Herbicides, glyphosate (Roundup) and S-metolachlor (DualMagnum), that have different modes of action compared to imidazolinone and ACCase herbicides will be used to screen the M2 plants cultivars Catahoula and Cocodrie. The Roundup herbicide will be applied on young seedlings with three to five leaves with the rate of 2 Qt/A. The S-Metolachlor herbicide will be applied folowing seed planting (pre-emergence) with the rate of 2 Pts/A (Dual Magnum).2. Production of mutated seed for herbicide screening: Mutation induction will be conducted annually for three years to produce adequate materials for mutation experiments. The concentration of mutagen ethyl methanesulfonate (EMS) and exposure duration will be determined based on small plot scale studies to avoid excessive mortality and/or high occurrences of phenotypic defects.3. Both F2 plants and F2:3 families from a cross between a herbicide-resistant line and non-resistant parent Cocodrie and/or Catahoula will be used in the inheritance study. The exact screening concentration of glyphosate will be determined in the lab to allow for the identification of both susceptible and resistant genotypes. Together with their appropriate parental lines, the F1s and F2s will be screened in herbicide solutions.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The results have been disseminated through national conferences (oral and poster presentations), annual reports, and international webinars. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this reporting period, the project provided special training for three scientists from Indonesia in the areas of high protein rice development. The training was funded by the Indonesian Atomic and Energy Agency, LIPI. In addition, theproject also provided professional training in the area of site-mutagenesis to knock-down two genes controlling the long day-length sensitivityusing a CRISPR/Cas9 system. The training was funded by theUSDA Borlaug Foundation. How have the results been disseminated to communities of interest?The results have been disseminated through national conferences (oral and poster presentations), field day presentation/posters and international webinars. What do you plan to do during the next reporting period to accomplish the goals? Continue to develop and evaluate advanced high-protein rice lines that have improved health benefits and grain quality to fulfill specific product requirements. Continue to develop and characterize new rice lines that are resistant to select classes of herbicides(ie glyphosate and S- metolachlor)to provide a wider choice of weed control and reduce costs.

Impacts
What was accomplished under these goals? Two additional promising advanced high-protein lines with improved yield were developed. Data from Preliminary Yield and Multi-Location trials are being compiled for these top lines to be used in consideration for release. Data from replicated Diversity PY tests evaluating promising lines that have high yield potential with improved protein contents ranging from 12-15% (w/w). A total of 20 new lines of high-protein rice developed had their yield potential improved by 11-17% compared to the yield of the first high-protein cultivar. The amylose contents range from 20 to 21% and milling from 60/68 to 63/71%, with an intermediate gelatinization temperature characteristic.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wenefrida, I. and H.S. Utomo. 2019. High protein rice and beyond. The 2019 ASA-CSSA-SSSA International Annual Meeting, Nov. 10-13, 2019, San Antonio, Texas
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Utomo, H.S. and I. Wenefrida. 2019. Breeding for high-protein rice. The 2019 ASA-CSSA-SSSA International Annual Meeting, Nov. 10-13, 2019, San Antonio, Texas
• Type: Other Status: Published Year Published: 2020 Citation: Wenefrida, I. and H.S. Utomo. 2020. Rice nutrition enhancement project: High protein line development and grain nutritional quality. The 111th Annual Research Report. H. Rouse Caffey Rice Research Station. Pp. 97-104.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Rice growers, rice industry, scientists, and academia Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results have been disseminated through presentations (oral and poster) in national conferences, as well as publications and rice field day posters. What do you plan to do during the next reporting period to accomplish the goals? Two top candidates of high-protein lines, Cocodrie derived and Francis derived, will be tested in Multi-Location trials at the H. Rouse Caffey Rice Research Station site and on-farm test sites in Vermilion, Evangeline, Acadia, and Jefferson Davis parishes. Twenty new promising lines will be evaluated in the Preliminary Yield trial at the H. Rouse Caffey Rice Research Station site using a randomized complete block design with cultural conditions similar to the ones for Multi-Location trials. Efforts to develop herbicide-resistant lines using M4 seeds that went through rigorous tests for S-Metolachlor resistance will continue. The field tests will be conducted in replicated plots in a randomized complete block design with increasing increment herbicide concentrations ranging from 1.0X (recommended lethal dose) to 3.0X (three times higher than) the recommended lethal dose.

Impacts
What was accomplished under these goals? Two promising advanced high-protein lines with improved yield were developed. Data from Preliminary Yield and Multi-Location trials are being compiled for these top lines to be used in consideration for release. Promising lines that have high yield and protein contents of 12-15% (w/w) were evaluated in replicated headrow plots as well as Preliminary Yield tests. Twenty new lines of high-protein rice developed had their yield potential improved by 11 to 17% compared to the yield of the first high-protein cultivar. Their amylose contents range from 20 to 21% and milling from 60/68 to 63/71%, with an intermediate gelatinization temperature characteristic.

Publications

• Type: Other Status: Published Year Published: 2019 Citation: Wenefrida, I. and H.S. Utomo. 2019. Rice nutrition enhancement project: High protein line development and grain nutritional quality. The 110th Annual Research Report. H. Rouse Caffey Rice Research Station. Pp. 97-104.

Progress 05/23/18 to 09/30/18

Outputs
Target Audience:Rice scientists, farmers/growers/extension agents, rice industry, and academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The resultshave been disseminated through publications, conference presentations, professional meetings, annual field day, and websites. What do you plan to do during the next reporting period to accomplish the goals?Next year's plan includes 1) continue develop and evaluate promising high-protein lines for potential new releases with improve health benefits and grain quality to fulfill specific product requirements and 2) continue develop and characterize new rice lines that are resistant to select classes of herbicides (ie glyphosate and S-metolachlor) to provide a wider choice of weed control.

Impacts
What was accomplished under these goals? The accomplishments during this reporting year include 1) Patent application to protect cultivar "Frontiere", 2) Promising new high-protein rice lines from different genetic background with higher protein content and improved yield potential, 3) M3 plant survivors from 0.75X of normal rate of Roundup (glyphosate) spay, and 4) M3 plant survivors from 2X of normal rate of Dual Magnum (S-metolachlor).

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wenefrida, I., H.S. Utomo, and D.E. Groth. 2018. Performance of high protein lines compared to the released cultivar Frontiere. The 37th RTWG (Rice Technical Working Group). Feb. 19-22, 2018, Long Beach, California. UC Davis.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Utomo, H.S. and I. Wenefrida. 2018. Sequence references of the amino acid transporter OsAAP6 found among high protein rice lines and field performance of marker-assisted breeding lines. The 37th RTWG (Rice Technical Working Group). Feb. 19-22, 2018, Long Beach, California. UC Davis.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2018 Citation: Wenefrida, I., H.S. Utomo, and D.E. Groth. 2018. Field performance of high protein rice lines. The 2018 ASA (American Society of Agronomy), CSSA (Crop Science Society of America), CSA (Canadian Society of Agronomy) Meeting.Baltimore Nov. 4-7, 2018.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2018 Citation: Utomo, H.S. and I. Wenefrida. 2018. Molecular signature of high protein rice lines. The 2018 ASA (American Society of Agronomy), CSSA (Crop Science Society of America), CSA (Canadian Society of Agronomy) Meeting. Baltimore Nov. 4-7, 2018.
• Type: Other Status: Published Year Published: 2018 Citation: Wenefrida, I., H.S. Utomo, and S.D. Linscombe. 2018. Rice Nutrition Enhancement Project: High protein line development and grain nutritional quality. The 109th Annual Research Report. H. Rouse Caffey Rice Research Station. Pp.105-110.
• Type: Other Status: Published Year Published: 2018 Citation: Wenefrida, I., H.S. Utomo, and D.E. Groth. 2018. RICE GRAIN NUTRITION ENHANCMENT PROJECT Introduction of Cahokia to Market, New High Potein Lines, and Herbicide Resistant Rice. Poster at the Annual Rice Field Day.