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Project Information


Reference Number 20180318
Project Title Functional validation of candidate genes to increase oil production in rice grain and leaf for nutrition, grain storage stability and disease resistance.
Project Type Contained Use (Laboratory)
Status Ongoing
Name of Institution International Rice Research Institute
Cooperating Institution Commonwealth Scientific and Industrial Research Organisation (CSIRO)
Supervising IBC International Rice Research Institute (IRRI)
Project Leader(s)

Inez H. Slamet-Loedin
Norman Oliva
Nese Sreenivasulu

Experimental Facility/Site Genetic Transformation Laboratory (GTL), Transgenic greenhouse and seed storage (Cl4)
Purpose / Objectives

1. To develop transgenic rice plants with increased leaf oil;

2. To develop transgenic rice plants with modified rice bran oil for longer shelf life and;

3. To develop transgenic rice plants with enhanced fungal disease resistance by overexpressing fatty acid modifying genes.

Biosafety measures

Restricted access and movement of transgenic materials. Storage of transgenic seeds in dedicated transgenic seed storage and proper disposition of excess transgenic materials

Conditions for Approval

a) All activities shall be conducted at the GTL and the CL4 Transgenic Glasshouse at IRRI, Los Baños, Laguna;

b) Thorough cleaning of vents and drainage of the CL4 Transgenic Glasshouse shall be conducted prior to the start of the activity;

c) The proponent shall submit to the DOST-BC a modified Gantt chart and shall adhere as close as possible to the revised schedule of activities;

d) Any modifications in the schedule and additional activities must be communicated to the DOST-BC for information and/or approval;

e) All confinement measures should be in place prior to the commencement of the activities;

f) Signage should be in place indicating that the facility is a restricted/limited access facility and only those personnel approved by the DOST-BC maybe allowed access;

g) Only authorized personnel as indicated in the proposal shall be allowed access to the facility for the entire duration of the experiment;

h) Monitoring for biosafety compliance shall be conducted by the Institutional Biosafety Committee (IBC), report of which shall be submitted to the DOST-BC within five (5) working days after the conduct of the monitoring activity;

i) Measures to limit access of unauthorized persons should be strictly observed during conduct of the experiment and that the designated facility must be kept secured at all times;

j) The DOST-BC shall be informed in advance of any visitations by unauthorized personnel for approval.

k) The DOST-BC shall be informed immediately of any intrusion by unauthorized persons;

l) The proponent shall ensure that stray animals are excluded from the facilities at all times;

m) In case of incident or undue destruction in the facility resulting from unauthorized entry of personnel or breach in containment, the proponent shall implement the biosafety contingency measures to prevent the inadvertent escape of any viable materials to the outside environment;

n) The proponent and the supervising IBC shall be held accountable for the undue destruction of the experimental materials and the consequences that their inadvertent escape may cause to the surrounding environment; and 

o) All viable materials within the experiment area shall be duly accounted for.

Date of Approval (DD-MM-YYYY) 19-01-2019
Date of Completion (DD-MM-YYYY) N/A
Executive Summary

Plants could be engineered in order to modify fatty acids that are nutritionally beneficial, with enhanced grain storage stability/shelf life and plant disease tolerance.

Plant-derived oils are one of the fastest growing agricultural commodities. World production of plant oils currently amounts to nearly 190 Mt with the vast majority being used for food and feed applications. Because non-seed tissues harbor a functional triacylglycerol (TAG) biosynthesis pathway and represent the majority of the above-ground plant biomass, engineering increased levels of TAG in vegetative organs has attracted increasing attention as a novel plant oil production system as shown by engineering of oilseed-like TAG levels in leaves of tobacco (Vanherckeet al., 2017). This was achieved by the simultaneous overexpression of WRINKLED1(WRI1), a positive regulator of fatty acid synthesis, acyl CoA:diacylglycerolacyl transferase 1 (DGAT1) which catalyses the final TAG assembly step, and Oleosin-L oil droplet protein. It was also observed that transcripts of the FATA thioesterase also increased.The first objective of this proposed contained experiment aims to develop transgenic rice plants with high leaf oil. Commonwealth Scientific and Industrial Research Organisation (CSIRO) will develop two constructs for overexpression of acyltransferases, transcription factor WRI1, Oleosin, and thioesterase genes, in combination to increase oil production in leaf. Genetic Transformation Laboratory (GTL) in IRRI will produce 20 to 30 events for each construct using Agrobacterium-based indica rice transformation with hygromycin selection.

The composition of rice (Oryza sativa L.) grain fatty acids (18% palmitic acid, 36% oleic acid and 37% linoleic acid) is not optimal for rice storage and utilization of rice bran oil as food grade oil. Genetic manipulation of fatty acid composition in rice bran oil to increase oleic acid levels at the expense of linoleic acid and palmitic acid would not only add extra value to the rice, but also enhance health benefits for consumers.   One of the major problems associated with the storage of brown rice and rice bran is that both rapidly deteriorate as the oil within them becomes rancid and produces stale flavors upon storage. Around 60–70% of rice bran oil currently produced is not fit for human consumption due to oxidative instability during storage of the bran. High levels of linoleic acid make rice bran oil prone to autoxidation. Rice bran oil also contains relatively high levels of palmitic acid. Palmitic acid is widely reported to raise total plasma cholesterol levels and is therefore detrimental to cardiovascular health.   In order to increase the value of rice bran oil, for human consumption it is desirable to increase the proportion of oleic acid at the expense of palmitic and linoleic acids using genetic engineering approach. RNA interference (RNAi) suppression of the expression of fatty acid desaturase, OsFAD2–1, has been shown to increase oleic acid and reduce linoleic and palmitic acids in rice grains. The second objective of this proposed contained experiment aims to develop transgenic rice with modified rice bran oil for longer shelf life. CSIRO will develop a vector for silencing of fatty acid desaturases gene(s) in grain for longer shelf life. GTL will produce 20 to 30 events for the construct using Agrobacterium-based indica rice transformation with hygromycin selection.

It has been known that fatty acids with specific functional group could be directly or indirectly involved in the biosynthesis of phytoalexin which give rise to resistance to numerous plant pathogens. In plants, the fatty acids are produced by expression of specific genes encoding fatty acid modifying enzymes which are involved in conversion of α-linolenic acid to the phytoalexin. This work is set up to test whether overexpressing such fatty acid modifying genes in rice could enhance the resistance to certain fungal diseases. The third objective of this proposed contained experiment aims to to enhance fungal disease resistance by overexpressing fatty acid modifying genes in transgenic rice. CSIRO will design two constructs for overexpression of fatty acid modifying genes, in combination for tolerance to fungal disease. GTL will produce 20 to 30 events for each constructs using Agrobacterium-based indica rice transformation with hygromycin selection. Screening for blast and some fungal diseases will be carried out in IRRI.

For all objectives, transgenic plants produced will be analyzed for transgenes presence. Furthermore, the number of inserts in positive plants will be determined by Southern blot. Expression studies of the selected plants using quantitative PCR will be made by GTL to determine transcript levels of the gene. The DNA or seeds derived from the selected primary transgenic lines will be sent to CSIRO for further analysis. All activities of the project will be conducted at GTL and the  transgenic glasshouse (CL4) at IRRI, Los Baños, Laguna. The project is expected to be completed in two years.