Project Information

Reference Number 20180314
Project Title 2018 lRRl Confined test of lron and Zinc biofortified transgenic rice
Project Type Confined Test
Status Ongoing
Name of Institution International Rice Research Institute
Cooperating Institution N/A
Supervising IBC International Rice Research Institute (IRRI)
Project Leader(s)

lnez H. Slamet-Loedin

Experimental Facility/Site N/A
Purpose / Objectives

The overall objective is to select three to four lead transgenic events with high dietary Fe and Zn (HIZR) in polished rice, with desirable agronomic performance, suitable for future multilocation trials.

The specific objectives are:

1. To evaluate the agronomic performance and ilron and Zinc trait of 8 HIZR events, selected from 11 events previously tested in the confined facility at lRRl in 2016 and 2017 (DOST-BC Ref No. 20164298) in two seasons (May- October 2018 and November -April 2019). The transgenic events contain either ferritin seed storage protein from soybean or non-soybean crops from Phaseo/us vulgaris, and apple (DOST-BC Ref No.2014-0281), and iron transporter/chelator gene from rice (DOST-BC Ref No. 2007-0221).

2. To compare the agronomic performance of the transgenic events with their respective recurrent parental cultivars and a popular check cultivar PSB Rc82.

3. To determine the lron and Zinc concentration in the polished grains.

4. To select the best among three (3) line-entries per event for future advancement.

Biosafety measures
  • The site is inside the TRF of the lRRl ZES. This area has been used previously for confined test of high lron and high Zinc biofortified materials (DOST-BC Ref No. 2014-0282 and 2016-0298) for four (4) seasons. lt is a gated facility.

  • 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 done by lRRl Experimental Farm or other contract workers, a regular, authorized personnel will be present to ensure adherence to biosafety protocols. Only DOSTBC approved visitors could access the site.

  • Temporal and physical isolation of the materials from the cultivated rice planted in nearby flelds will be observed.

  • Transgenic material handling such as transfer and disposal will be done in accordance to the guidleline set by lRRl's Biosafety Office, the IBC and BPI-PEQS as monitoring arm of the DOST-BC.

Conditions for Approval

a) Activities shall be conducted at the HIZR confined test area inside the Transgenic Research Facility (TRF) of Zeigler Experimental Station (ZES) at the International Rice Research Institute (IRRI), Los Banos, Laguna.

b) 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 .

c) 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.

d) 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 conducted.

e) 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.

f) A letter will be issued to the IBC informing that planting/transplanting on the confined test site can commence.

g) The proponent shall adhere as closely as possible to the schedule of implementation and monitoring activities for this activity.

h) Modifications in the planting schedule and other activities must be communicated to the DOST-BC for information and/or approval.

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

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

k) All confinement measures should be in place before the actual transplanting of rice seedlings.

l) The proponent shall ensure that only authorized personnel are allowed inside the test site.

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

n) The DOST-BC shall be informed immediately of any intrusion by unauthorized persons.

o) 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.

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 facility, the proponent should implement measures to prevent the inadvertent escape of any viable material within the site.

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 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 experiment period.

y) The proponent shall submit through the IBC a completion report ninety (90) days after the official completion of the confined test after completion of all post-harvest monitoring of volunteers.

Date of Approval (DD-MM-YYYY) 24-03-2018
Date of Completion (DD-MM-YYYY) N/A
Executive Summary

lron (Fe) andZinc (Zn) deficiencies are the most common micronutrient deficiencies globally affecting more than 2 billion people. Biofortification of rice to increase micronutrient content, particularly Fe and
Zn, 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, hence they are not available for absorption by the
human body. Polished (milled). rice grains of com.monly cultivated and consumed rice varieties only contain approximately 2 pS g.1 Fe and 16 trg g'1 Zn. The HarvestPlus rice breeding programs for
biofortified rice target 13 t-rg g- Fe and 28 pS g' Zn in milled rice grains to reach approximately 30% of the estimated average requirement (EAR) in human (Bouis et a|.,2011 ). The increase of grain Zn
was made possible because the inserted rice gene resulted in the chelation and translocation to the grains of both Fe and Zn, but not the undesirable heavy metals (i.e. Cadmium or arsenic). The
transgenic approach was pursued because conventionally-bred rice lines could only reach a maximum of 7-8 pg g-' Fe and 24 ttg g't Zn in polished rice grains.

We developed transgenic indica rice lines (using cultivars lR64 and 1R69428 as backgrounds) harboring iron storage protein ferritin gene from soybean (NCBP Ref. No. 1999-01 19) and iron
transporter/chelator genes from rice (NCBP Ref No. 2007-0221). One event was identified to have exceeded the nutritionaltarget levelsfor Fe and Zn in polished giains (14-15 Hg g'1 and 4548 pg g'1,
respectively). This event (NASFer-274 or lRS495-274) had no statistically significant yield reduction when evaluated under confined test conditions (DOST-BC Ref no. 2011-0264, DOST-BC Ref no.
2013-0276, DOST-BC Ref No.20'14-0282) (Trijatmiko et al., 2016). 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 Rc238) and BRRI Dhan 28 have also been developed (DOST-BC Ref. No. 2014-0281). Events with Fe concentration in the polished grains of more thanl0
1tg g'' and Zn concentration of more than 30 prg g-t have been evaluated for two seasons in confined test (DOST-BC Ref No.2016-0298).

This proposed confined test aims to evaluate and select three to four candidate lead events (from eight independent events) with high dietary Fe and Zn (HIZR) in polished rice grains and with
desirable agronomic performance, ln 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 and four (4) check varieties - NSIC Rc238, PSBRc82, BRRI Dhan 28, BRRI Dhan 29 (2 lines per check vaieties per replicate) seryes as controls. The phenotyping will include the
agronomic characteristics and the polished grain Fe and Zn concentrations.

The site of the confined test is the confined test area designated for High lron and Zinc rice inside the Transgenic Research Facility (TRF) of Zeigler Experimental Station (ZES) at the lnternational Rice
Research lnstitute (lRRl), Los Bafros, Laguna. The duration of the confined test is from April 2018 to April 2019. Harvested seeds will be kept in hermetically-sealed aluminum foil packages and stored in
the storage area designated for transgenic seeds at lRRl. Other materials or excess seeds may be discarded following existing lRRl biosafety protocols.