Dr. Reynante L. Ordonio

1. To evaluate the agronomic performance and grain Fe and Zn concentrations of promising transgenic events containing genes encoding for iron storage-protein from dicot crops [soybean, kidney bean and apple] (DOST-BC: 2014-0281), and for iron transporter/chelator from rice (DOST-BC: 2007-0221) in BR28, NSIC Rc238 and BR28 genetic background in a replicated yield trial.

2. To identify and select lines and events for advancement in the next generation.

• The site is inside the indentified Confined Test Area of the PHILRICE Batac Station. This area has been used previously for confined test of Golden Rice GR2E event materials (DOST-BC: 2015-0292) for two seasons.
• The confined test area will be accessed only by the authorized staff who are directly involved in the experiment (Annex 9). For additional labor and activities which will be done during the conduct of the experiment, a regular, authorized personnel will be present to ensure adherence to biosafety protocols. Only DOST-BC approved emergency laborers/visitors could access the site.
• Temporal and physical isolation of the materials from the cultivated rice planted in nearby fields will be observed.

a) Activities shall be conducted at the transgenic test facility inside the experimental field of the PhilRice-Batac Station in Brgy. Tabug, Batac City, Ilocos Norte and shall be supervised by the PHILRICE-Batac-IBC;

b) The DOST-BC approved Project Information Sheet (PIS) shall be posted for three (3) consecutive weeks in the public announcement board of the barangay(s) and city or municipality where the proposed activities will be carried out;

c) Proof of posting of the PIS, which may either be a certification from the duly authorized Barangay leader(s) or an affidavit executed by the Proponent, should be submitted to the DOST-BC within ten (10) days from the last day of posting;

d) Only upon completion of the public comment period and determination that no comments were received or comments were satisfactorily addressed by the proponent shall the DOST-BC issue a letter to the PhilRice-Batac-IBC informing that planting/transplanting on the confined test site can commence;

e) Prior to the initiation of the activities, the proponents shall discuss the monitoring schedule with the Department of Agriculture-Bureau of Plant Industry-National Plant Quarantine Service Division (DA­BPl-NPQSD) to identify the activities that will require their presence. The proponents shall furnish the DOST-BC with a copy of the agreed monitoring schedule;

f) All confinement measures should be in place before the actual transplanting of rice seedlings;

g) At least 150 meters physical or temporal reproductive isolation of the GMO shall be observed at all times;

h) The proponent shall ensure that rodent control measures are in place and that stray animals are excluded from the test site while trials are being conducted;

i) The proponent shall adhere as closely as possible to the schedule of implementation and monitoring activities for this project;

j) Modifications in the planting schedule and other activities must be officially communicated by the PHILRICE-Batac-IBC to the DOST-BC for information and/or approval;

k) Monitoring for biosafety compliance during stages of the project implementation, such as, but not limited to, the movement of transgenic materials, transplanting, reproductive stage, harvest, post­harvest monitoring and termination, shall be conducted by the IBC. Report of which shall be submitted to the DOST-BC not more than five (5) days after the conduct of the monitoring activity;

l) Movement of all transgenic materials should be done in compliance with the DOST-BC and the DA-BPl-NPQSD guidelines;

m) The proponent shall ensure that only authorized personnel are allowed inside the confined test site;

n) The proponent shall inform the DOST-BC in advance of any visitations by unauthorized personnel, for approval;

o) The proponent shall inform the DOST-BC immediately of any intrusion by unauthorized persons;

p) The proponent shall ensure the security of the trial, the field workers, and the monitors;

q) In case of undue destruction of the experimental materials resulting from unauthorized entry of personnel or breach of containment of the confined test site, the proponent should implement measures to prevent the inadvertent escape of any viable material to the outside environment;

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

s) The rice grains or any plant part shall not be eaten or fed to humans, wildlife and livestock;

t) The proponent shall provide a space within the test site for disposal of the used plant materials;

u) The harvested grains shall be properly labeled and stored at the designated seed storage facility where transgenic seeds are kept, while plant materials and other plant debris used within the test site should be buried in a pit within the test site or plowed under;

v) The proponent shall leave the test site fallow for at least thirty (30) days. During the fallow period, the proponent shall strictly monitor emergence of volunteer plants and plant pests/diseases within the site for two (2) consecutive weeks after harvest. If necessary, the proponent may irrigate the test site to induce germination of seeds. Any volunteer plants that will emerge, or any ratoon residues shall be uprooted and disposed either by plowing under or by burying in the pit intended for transgenic plant biomass from this confined field test;

w) The IBC shall determine whether the confined test site shall be released from post-harvest restrictions. This shall form part of the collated post-harvest monitoring report of the project to be submitted IBC;

x) The proponent shall comply with additional requirements that the DOST-BC may impose, as necessary, during the conduct of the experiment;

y) The proponent shall submit, through the IBC, a completion report within ninety (90) days after the official completion of the confined test and after all post harvest activities have been completed; and

z) The PHILRICE-Batac-IBC shall submit a report on all its monitoring activities upon completion of this project by submitting an IBC completion report in the prescribed format.

Iron (Fe) and zinc (Zn) deficiencies are the most common micronutrient deficiencies globally affecting more than 2 billion people. Biofortification of rice to increase micronutrient content, particularly iron, is considered one of the options to complement existing approaches to alleviate this ‘hidden hunger’. Rice grains of commonly cultivated varieties contain low levels of Fe when milled, while in brown rice, Fe and Zn in the bran are mostly bound to phytate, and less available for absorption by the human body. Polished (milled) rice grains of commonly cultivated and consumed rice varieties only contain approximately 2 ppm Fe and 16 ppm Zn. The HarvestPlus rice breeding programs for biofortified rice target 13 ppm Fe and 28 ppm Zn in milled rice grains to reach approximately 30% of the estimated average requirement (EAR) in human (Bouis et al., 2011). The increase of grain Zn was made possible because the inserted rice gene proved to chelate and translocate to the grains both Fe and Zn, but not the undesirable heavy metals (i.e. cadmium or arsenic). Transgenic approach was pursued as the target levels for Fe and Zn cannot be reached through conventional breeding. The highest amount recorded for Zn in well-polished grains of conventionally bred lines is 24 ppm while 5 ppm for Fe.

IRRI developed transgenic indica rice lines (using cultivars IR64 and IR69428 as backgrounds) harboring iron storage protein ferritin gene from soybean (NCBP Ref. no. 1999-0119) and iron transporter/chelator genes from rice (NCBP Ref no. 2007-0221). One event was identified exceeding the nutritional target levels for Fe and Zn in well-polished grains (14-15 ppm Fe and 45-48 ppm Zn). This event (IRS495-274) had no statistically significant yield reduction when evaluated under confined tests (DOST-BC Ref no. 2011-0264, DOST-BC Ref no. 2013-0276, DOST-BC Ref no. No. 2014-0282) (Trijatmiko et al., 2016). Since the published report (Trijatmiko et al., 2016) IRRI have developed a set of back-up events using dicot ferritins genes in combination with rice iron homeostasis genes or solely rice genes in the background varieties of IRRI 156 (NSIC Rc 238) and BRRI Dhan 28 (DOST-BC ref. no. 2014-0281). Initially, 11 back-up events were selected based on assessments in the transgenic screen house. Events with Fe concentration in the polished grains >10 ppm and Zn concentration > 30 ppm have been evaluated for two seasons in confined test (DOST-BC Ref no. 2016-0298).

This proposed confined test is aimed to evaluate and select three to four candidate lead events with high dietary Fe and Zn (HIZR) in well-polished rice and desirable agronomic performance from eight independent events. In total 32 entries will be evaluated in 3 replications. The 32 entries consist of eight (8) independent events (3 lines per event) selected from 11 events tested in the last confined test done at the IRRI Zeigler Experimental Station in Aug-Dec 2017. Four (4) check varieties (2 lines per check varieties) are also included to serve as controls, namely: NSIC Rc 238, PSBRc82, BRRI Dhan 28, and BRRI Dhan 29. The phenotyping will include the agronomic characteristics and the well-polished grain Fe and Zn concentrations.

The confined test will be conducted in the transgenic test facility inside the experimental field of the PhilRice-Batac Station in Batac City, Ilocos Norte. The duration of the confined test is from June 2018 to July 2019.